CN116173297A

CN116173297A - Improvements in methods for immobilizing biological materials

Info

Publication number: CN116173297A
Application number: CN202310010152.9A
Authority: CN
Inventors: P·安东尼; M·埃里克森; A·盖尔黑根; E·科赫; D·尼斯特罗姆; C·波施-格拉姆; H·戈兰森
Original assignee: Carmeda AB
Current assignee: Carmeda AB
Priority date: 2018-03-09
Filing date: 2019-03-08
Publication date: 2023-05-30
Also published as: AU2019232163A2; CN111867647A; CN111867647B; CA3093027A1; SG11202008063YA; KR20200129114A; JP2021514881A; AU2019232163A1; US20190275212A1; ES2953184T3; JP7365371B2; US20220378985A1; EP3762055A1; WO2019170858A1; EP3762055B1; BR112020017412A2

Abstract

The present invention relates to improvements in methods for immobilizing biological materials. The present invention provides a method of preparing a solid object having a surface comprising a layered coating of a cationic and anionic polymer, wherein the outer coating comprises an anticoagulant substance, the method comprising the steps of: i) Treating the surface of the solid object with a cationic polymer; ii) treating the surface with an anionic polymer; iii) Optionally repeating steps i) and ii) one or more times; iv) treating the surface with a cationic polymer; and v) treating the outermost layer of the cationic polymer with an anticoagulant substance, whereby the anticoagulant substance is covalently linked to the outermost layer of the cationic polymer; wherein the anionic polymer is characterized by having: (a) a total molecular weight of 650kDa to 10,000 kDa; and (b) a solution charge density of >4 μeq/g; and wherein step ii) is carried out at a salt concentration of 0.25M to 5.0M.

Description

Improvements in methods for immobilizing biological materials

The present application is a divisional application of PCT application PCT/EP2019/055845, entitled "improvement of method for immobilizing biological substances" filed on 3/8 of 2019, which enters china with a stage date of 2020, 9/201980018210.4.

Technical Field

The invention relates to a method for producing a solid object having a surface coating containing biological substances (substances). In particular, the present invention relates to a process for preparing an improved surface coating comprising an anticoagulant substance such as heparin and certain products resulting therefrom.

Background

When a medical device is implanted in the body or in contact with body fluids, many different reactions start to act, some of which cause inflammation and some of which cause blood contacting the surface of the device to coagulate. To counteract these serious adverse effects, the well-known anticoagulant compound heparin is administered systemically to patients for a long period of time before or while the medical device is implanted in the body or when it is in contact with body fluids, in order to provide antithrombotic effects.

Thrombin is one of several coagulation factors, all of which work together to cause thrombosis of the surface in contact with blood. Antithrombin (also known as antithrombin III) ("ATIII") is the most predominant coagulation inhibitor. It counteracts the effects of thrombin and other coagulation factors, thereby restricting or limiting blood clotting. Heparin significantly increases the rate at which antithrombin inhibits clotting factors. Heparin cofactor II ("HCII") is another coagulation factor that rapidly inhibits thrombin in the presence of heparin.

However, systemic treatment with high doses of heparin is often accompanied by serious side effects, with bleeding being predominant. Another rare but serious complication of heparin therapy is the development of an allergic reaction called heparin-induced thrombocytopenia (HIT), which can lead to thrombosis (both venous and arterial). High dose systemic heparin therapy (e.g., during surgery) also requires frequent monitoring of activated clotting time (for monitoring and guiding heparin therapy) and corresponding dose adjustments when needed.

Thus, solutions have been sought in cases where systemic heparinization of the patient is not necessary or possible to be limited. It is believed that this can be achieved by surface modification of the medical device by exploiting the anticoagulant properties of heparin and other anticoagulants. Thus, a number of more or less successful techniques have been developed in which a layer of heparin is attached to the surface of the medical device, thereby seeking to render the surface antithrombotic. For devices requiring long-term biological activity, heparin should ideally have resistance to leaching and degradation.

Heparin is a polysaccharide that carries negatively charged sulfate and carboxylic acid groups on the saccharide units. Ionic binding of heparin to the polycationic surface was thus attempted, but the surface modification lacks stability due to heparin leaching from the surface resulting in loss of function. Thereafter, different surface modifications were prepared in which heparin had been covalently bound to groups on the surface.

One of the most successful methods of rendering medical devices thromboresistant is to covalently bind heparin fragments to the modified surface of the device. General methods and their modifications are described in various patent documents (see EP 008686 A1, EP0086187A1, EP0495820B1 and US6,461,665B1, all of which are incorporated herein by reference in their entirety).

These documents describe the preparation of heparinized surfaces by reacting heparin modified to bear terminal aldehyde groups with the surface of medical devices modified to bear primary amino groups. An intermediate schiff base is formed which is reduced in situ to form a stable secondary amine linking group, thereby covalently immobilizing heparin.

Other methods of covalently attaching heparin to a surface while maintaining its activity are described in WO2010/029189A2, WO2011/110684A1, and WO2012/123384A1 (the entire contents of which are incorporated herein by reference).

The anticoagulant substance is typically immobilized on a surface that has been treated with one or more layers of polymer or complex, rather than directly on the surface of a solid object.

EP0086187A1 describes a surface-modified substrate onto which a complex is adsorbed, wherein the complex has a polymeric cationic surfactant comprising a primary amino nitrogen function and a secondary and/or tertiary amino function and a dialdehyde having 1 to 4 carbon atoms between the two aldehyde groups. The anionic compound may additionally be bonded to the complex and optionally additional cationic and anionic alternating compounds.

EP0495820B1 describes a method of modifying a substrate surface, the method comprising the steps of: (a) Adsorbing a polyamine having a high average molecular weight and crosslinking the polyamine with crotonaldehyde; (b) Then adsorbing a layer of anionic polysaccharide on the surface of the crosslinked polyamine; (c) Optionally repeating steps (a) and (b) one or more times; and (d) adsorbing a layer of uncrosslinked polyamine providing free primary amino groups on the anionic polysaccharide layer or the outermost layer of anionic polysaccharide. In a subsequent step, a biologically active chemical species bearing a functional group that reacts with the free primary amino group may be combined with an uncrosslinked polyamine, such as heparin.

However, there remains a need for improved surface coatings comprising anticoagulant substances, such as heparin, in particular coatings wherein the biological activity of the anticoagulant substances is maintained or enhanced. Such improved surface coatings have potential utility in medical devices and other articles that would benefit from anticoagulated surfaces.

The inventors have found that, surprisingly, the properties of the surface on which the anticoagulant substance is immobilized can significantly influence the properties of the coating, in particular the biological activity of the resulting anticoagulant substance. In particular, when the anticoagulant substance is immobilized on the surface of a solid object comprising a layered coating of cationic and anionic polymers, careful adjustment of the properties and application conditions of the anionic polymer layer may improve the properties of the resulting solid object coating, including for example the antithrombotic properties it may have.

Disclosure of Invention

In one aspect, the present invention provides a method for preparing a solid object having a surface comprising a layered coating of cationic and anionic polymers, wherein the outer coating comprises an anticoagulant substance, the method comprising the steps of:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

v) treating the outermost layer of the cationic polymer with an anticoagulant substance, whereby the anticoagulant substance is covalently linked to the outermost layer of the cationic polymer;

wherein, the liquid crystal display device comprises a liquid crystal display device,

the anionic polymer is characterized by having: (a) a total molecular weight of 650kDa to 10,000 kDa; and (b) a solution charge density of >4 μeq/g;

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

In another aspect, the present invention provides a method for preparing a solid object having a surface comprising a layered coating of cationic and anionic polymers, wherein the outer coating comprises an anticoagulant substance, the method comprising the steps of:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

the anionic polymer is a polymer comprising-SO ₃ ^- A polymer of the group(s),

the anionic polymer is characterized by having: (a) a total molecular weight of 650kDa to 10,000 kDa; and (b) a sulfur content of 10% to 25% by weight of the anionic polymer;

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

the anionic polymer is characterized by having a total molecular weight of 650kDa to 10,000 kDa;

the anionic polymer is dextran sulfate;

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

Drawings

FIG. 1: showing an embodiment of the coating of the present invention having a single bilayer;

FIG. 2: shows normalized Heparin Activity (HA) of PVC tubes coated with

dextran sulfate

4, 5, 6 and 7 at 0.25M and 1.7M NaCl concentrations;

FIG. 3: shows standardized Heparin Activity (HA) of PVC pipes coated with dextran sulfate 5, with different salts applied at different concentrations;

FIG. 4: heparin Concentration (HC) of PVC tubes coated with

dextran sulfate

3, 4, 5, 6 and 7 at 1.7M NaCl concentration;

FIG. 5: heparin Concentration (HC) of PVC pipes coated with

dextran sulfate

3, 4, 5, 6 and 7 at different NaCl concentrations are shown;

FIG. 6: heparin Concentration (HC) of PVC pipes coated with dextran sulfate 5, with different salts applied at different concentrations;

FIG. 7: the zeta potential of PVC pipes coated with

dextran sulfate

3, 4 and 5 at a concentration of 1.7M NaCl is shown;

FIG. 8: the zeta potential of PVC pipes coated with

dextran sulfate

3, 6 and 7 at a concentration of 1.7M NaCl is shown;

FIG. 9: the zeta potential of PVC pipes coated with

dextran sulfate

3, 4 and 5 at 0.25M NaCl concentration is shown;

FIG. 10: the zeta potential of PVC pipes coated with

dextran sulfate

3, 6 and 7 at 0.25M NaCl concentration is shown;

FIG. 11: the zeta potential of PVC pipes coated with dextran sulfate 5 at different NaCl concentrations is shown;

FIG. 12: dextran sulfate 5 was shown to be at different Na ₂ HPO ₄ Zeta potential of the concentration coated PVC pipe;

FIG. 13: dextran sulfate 5 was shown to be at different Na ₂ SO ₄ Zeta potential of the concentration coated PVC pipe;

FIG. 14: preserved platelets (%) showing PVC tubes coated with

dextran sulfate

2, 4, 5, 6 and 7 at 0.25M NaCl concentration;

FIG. 15: f1+2 (prothrombin fragments) showing PVC tubes coated with

dextran sulfate

2, 4, 5, 6 and 7 at 0.25M NaCl concentration;

FIG. 16: preserved platelets (%) showing PVC tubes coated with

dextran sulfate

4, 5, 6 and 7 at a concentration of 1.7M NaCl;

FIG. 17: f1+2 (prothrombin fragment) of PVC tubes coated with

dextran sulfate

4, 5, 6 and 7 at 1.7M NaCl concentration is shown;

FIG. 18: preserved platelets (%) showing PVC tubes coated with dextran sulfate 4 at 0.25M NaCl concentration before and after temperature and humidity testing;

FIG. 19: f1+2 (prothrombin fragment) showing PVC tubes coated with dextran sulfate 4 at 0.25M NaCl concentration before and after temperature and humidity testing;

FIG. 20: preserved platelets (%) showing PVC tubes coated with

dextran sulfate

4, 5 and 7 at 1.7MNaCl concentration before and after temperature and humidity testing;

FIG. 21: f1+2 (prothrombin fragments) showing PVC tubes coated with

dextran sulfate

fig. 22: showing a typical zeta potential profile of the solid object of the present invention.

FIG. 23: shows the active pentasaccharide sequence of heparin.

Detailed Description

Solid object

Any solid object can potentially be coated using the methods of the present invention, however, such coatings and methods are particularly useful for medical devices, analytical devices, separation devices, and other industrial articles including membranes.

The solid object may have an antithrombotic surface. In certain embodiments of the invention, the antithrombotic surface may exhibit a direct pharmacological inhibition of the coagulation response by immobilizing an anticoagulant substance. In certain embodiments of the invention, the antithrombotic surface does not cause any significant clinically significant adverse effects upon contact with blood, such as thrombosis, hemolysis, platelet, leukocyte and complement activation, and/or other blood-related adverse effects.

In the art, the terms "hemocompatible", "non-thrombotic", "antithrombotic" and the like are generally to be interpreted as equivalent to the term "antithrombotic".

In one embodiment, the solid is a medical device. When the solid object is a medical device, it is suitably an antithrombotic medical device. Thus, in one embodiment, the solid object is an antithrombotic medical device. For the purposes of this patent application, the term "medical device" refers to an in vivo or an in vitro device, but more generally refers to an in vivo medical device.

An in vivo medical device is a device used within an anatomical structure, such as within the vasculature or other body lumen, space, or cavity, which typically may provide a therapeutic effect. The in vivo device may have long-term or temporary use. Immediately after the surgical procedure, the long-term used devices are left partially or fully in the anatomy to deliver them, e.g., stents or stent-grafts. Temporary or short-term use devices include those that are temporarily inserted into the treatment area (i.e., inserted and then removed in the same surgical procedure), such as medical balloons. In one embodiment, the solid object is an in vivo medical device.

Examples of in vivo medical devices, which may be permanent or temporary, include stents, including bifurcated stents, balloon-expandable stents, self-expanding stents, neurovascular stents, and shunt stents, stent-grafts, including bifurcated stent-grafts, including vascular grafts and bifurcated grafts, sheaths, including retractable sheaths, such as interventional diagnostic and therapeutic sheaths, large and standard bore endovascular delivery sheaths, arterial introducer sheaths with or without hemostatic control, and arterial introducer sheaths with or without steering, micro-introducer sheaths, dialysis access sheaths, introducer sheaths, and percutaneous sheaths, dilators, occluders, such as vascular occluders, embolic filters, embolic ablation devices, catheters, vascular grafts, blood indwelling monitoring devices; valves, including prosthetic heart valves, pacer electrodes, guidewires, cardiac leads, cardiopulmonary bypass circuits, cannulas, plugs, drug delivery devices, balloons, tissue patch devices, blood pumps, patches, lines, such as long-term infusion lines or arterial lines, placement lines, devices for continuous subarachnoid infusion, feeding tubes, CNS shunts, such as ventricular thoracic shunts, ventricular Atrial (VA) shunts, ventricular abdominal (VP) shunts, ventricular shunts, portal shunts, and shunts for ascites.

Examples of catheters (catheters) include, but are not limited to, microcatheters, central venous catheters, peripheral intravenous catheters, hemodialysis catheters, catheters such as coated catheters, including implantable venous catheters, fenestrated venous catheters, ultrasonically operated coronary catheters for angiography, angioplasty, or in the heart or peripheral veins and arteries, catheters containing spectroscopic or imaging capabilities, hepatic arterial infusion catheters, CVCs (central venous catheters), peripheral intravenous catheters, peripherally inserted central venous catheters (PIC lines), flow-directed balloon-tipped pulmonary arterial catheters, parenteral total nutrient catheters, long-term indwelling catheters (e.g., long-term indwelling gastrointestinal catheters and long-term indwelling urogenital catheters), peritoneal dialysis catheters, CPB catheters (cardiopulmonary bypass), urinary catheters and microcatheters (e.g., for intracranial applications).

In one embodiment, the solid object is an in vivo medical device selected from the group consisting of a stent, a stent-graft, a sheath, a dilator, an occluder, a valve, an embolic filter, an embolic removal device, a catheter, an artificial blood vessel, a blood retention monitoring device, a valve, a pacer electrode, a lead, a cardiac lead, a cardiopulmonary bypass circuit, a cannula, a plug, a drug delivery device, a balloon, a tissue patch device, a blood pump, a patch, a line, a placement wire, a device for continuous infusion of a subarachnoid space, a feeding tube, and a shunt. In a specific embodiment, the solid object is a stent or stent-graft.

In one embodiment, the in vivo medical device may be used for neurological, peripheral, cardiac, orthopedic, skin or gynecological applications. In one embodiment, the scaffold may be used for cardiac, peripheral or nervous system applications. In one embodiment, the stent-graft may be used for cardiac, peripheral or nervous system applications. In one embodiment, the sheath may be used for carotid, renal, transradial, transseptal, pediatric, or microscopic applications.

Examples of extracorporeal medical apparatus are blood processing apparatus and blood transfusion apparatus. In one embodiment, the in vivo medical device may be used for neurological, peripheral, cardiac, orthopedic, skin or gynecological applications. In one embodiment, the extracorporeal medical apparatus is an oxygenator. In another embodiment, the in vitro medical device is a filter capable of removing viruses, bacteria, pro-inflammatory cytokines and toxins that cause sepsis.

For example, the membrane may be a hemodialysis membrane.

For example, the analytical device may be a solid support for performing analytical methods, such as chromatography or immunological assays, reaction chemistry or catalysis. Examples of such devices include slides, beads, well plates, and membranes.

For example, the separation device may be a solid support for performing a separation method, such as protein purification, affinity chromatography or ion exchange. Examples of such devices include filters and columns.

The solid body may comprise or consist of, in particular, a metal, a synthetic or naturally occurring organic or inorganic polymer, a ceramic material, a protein-based material or a polysaccharide-based material.

Suitable metals include, but are not limited to, biocompatible metals such as titanium, stainless steel, high nitrogen stainless steel, cobalt, chromium, nickel, tantalum, niobium, gold, silver, rhodium, zinc, platinum, rubidium, copper, and magnesium, and combinations (alloys) thereof. Suitable alloys include cobalt-chromium alloys such as L-605, MP35N, elgiloy, titanium alloys including nickel-titanium alloys (e.g., nitinol), tantalum alloys, niobium alloys (e.g., nb-1% Zr), and the like.

In one embodiment, the biocompatible metal is a nickel-titanium alloy, such as nitinol.

Synthetic or naturally occurring organic or inorganic polymers include polyolefins, polyesters (e.g., polyethylene terephthalate and polybutylene terephthalate), polyester ethers, polyester elastomer copolymers (e.g., those commercially available under the trade name hytrel. Rtm from dupont of Wilmington, del.), fluoropolymers, chlorine-containing polymers (e.g., polyvinyl chloride (PVC)), block copolymer elastomers (e.g., those having styrene end blocks and intermediate blocks formed from butadiene, isoprene, ethylene/butylene, ethylene/propylene), block copolymers (e.g., styrene block copolymers such as acrylonitrile-styrene and acrylonitrile-butadiene-styrene block copolymers, or block copolymers wherein the particular block copolymer is a thermoplastic elastomer, wherein the block copolymer is comprised of hard segments of polyester or polyamide and soft segments of polyether), polyurethanes, polyamides (e.g., nylon 12, nylon 11, nylon 9, nylon 6/9, and nylon 6/6), polyether block amides (e.g., such as nylon 6/6)

) Polyether ester amides, polyimides, polycarbonates, polyphenylene sulfides, polyphenylene oxides, polyethers, silicones, polycarbonates, polyhydroxyethyl methacrylates, polyvinylpyrrolidone, polyvinyl alcohols, rubbers, silicone rubbers, polyhydroxy acids, polyallylamines, polyacrylamides, polyacrylic acids, polystyrenes, polytetrafluoroethylene, polymethyl methacrylates, polyacrylonitrile, poly (vinyl acetate), poly (vinyl alcohol), polyoxymethylene, polycarbonates, phenolics, amino epoxy resins, cellulose-based plastics, and rubber-like plastics, bioabsorbableSex substances such as poly (D, L-lactide) and glycolipids, and copolymers thereof, derivatives thereof, and mixtures thereof. Combinations of these materials can be used with and without crosslinking. Some of these classes can be used as both thermoset materials and thermoplastic polymers. As used herein, the term "copolymer" will be used to refer to any polymer formed from two or more monomers, e.g., 2, 3, 4, 5, etc.

Fluorinated polymers (fluoropolymers) include fluoropolymers such as expanded polytetrafluoroethylene (ePTFE), polytetrafluoroethylene (PTFE), fluorinated ethylene-propylene (FEP), perfluorocarbon copolymers such as tetrafluoroethylene perfluoroalkyl vinyl ether (TFE/PAVE) copolymers and copolymers of Tetrafluoroethylene (TFE) and perfluoromethyl vinyl ether (PMVE), and combinations of the foregoing with and without cross-links between polymer chains.

In one embodiment, the solid body comprises a polyether-block-amide such as

In another embodiment, the solid object comprises a chlorine-containing polymer (e.g., PVC) or a fluoropolymer (e.g., ePTFE).

The polymeric substrate may optionally be blended with fillers and/or colorants. Thus, suitable substrates include colored materials, such as colored polymeric materials.

Ceramic substrates may include, but are not limited to, silica, alumina, silica, hydroxyapatite, glass, calcium oxide, polysilanol, and phosphorous oxide.

Protein-based materials include silk and wool. Polysaccharide-based materials include agarose and alginate.

Anticoagulation material (anticoagulant entity)

An anticoagulant substance is a substance that is capable of interacting with mammalian blood to prevent or reduce coagulation or thrombosis.

The anticoagulant substance includes: heparin fraction, dermatan sulfate fraction, dermatan disulfate fraction, hirudin, eptifibatide, tirofiban (tirofiban), urokinase, D-Phe-Pro-Arg chloromethylketone, RGD peptide-containing compounds, AZX100 (cell peptide mimicking HSP20, capstone Therapeutics company, USA), platelet receptor antagonists, antithrombin antibodies, anti-platelet receptor antibodies, aspirin, prostaglandin inhibitors, platelet inhibitors (e.g. clopidogrel, nitric Oxide (NO), prostaglandins and acipimab), anti-platelet peptides, coumarins (i.e. 4-hydroxycoumarins vitamin K antagonists such as warfarin), argatroban, thrombomodulin, anticoagulants (e.g. apyrase). In one embodiment, the anticoagulant substance is selected from the group consisting of heparin fraction, dermatan sulfate fraction, and dermatan disulfide fraction.

In one embodiment, the anticoagulant substance is a glycosaminoglycan. In one embodiment, the anticoagulant substance is a thrombin inhibitor.

The term "heparin moiety" refers to a heparin molecule, a fragment of a heparin molecule, a derivative of a heparin molecule, or an analog of a heparin molecule.

In one embodiment, the anticoagulant substance is a heparin moiety. Suitably, the heparin moiety is selected from: full length heparin (natural heparin), alkali or alkaline earth metal salts of heparin (e.g., heparin sodium (e.g., hepsal or pullarin), heparin potassium (e.g., clarin), heparin lithium, heparin calcium (e.g., calciparine) or heparin magnesium (e.g., cuthesparine)), low molecular weight heparin (e.g., adequan sodium, tinzaparin sodium or dalteparine sodium), heparan sulfate, heparan, heparin-based compounds, heparin with hydrophobic counter ions, synthetic heparin compositions capable of inhibiting factor Xa action by antithrombin (e.g., a "fondaparinux sodium" composition (e.g., arixta from the company glazink)), and synthetic heparin derivatives comprising at least an active pentasaccharide sequence from heparin (see, e.g., petitou et al, biochimie,2003,85 (1-2): 83-9). Other heparin moieties include heparin modified by, for example, moderate nitrous acid degradation (US4,613,665A, incorporated herein by reference in its entirety) or periodate oxidation (US6,653,457B1, incorporated herein by reference in its entirety) and other modification reactions known in the art, wherein the activity of the heparin moiety is preserved. Heparin moieties also include moieties that bind to a linking group or spacer group as described below. In one embodiment, the heparin moiety is full length heparin.

For example, low molecular weight heparin may be prepared by oxidative depolymerization, enzymatic degradation, or deamination cleavage.

In one embodiment, the heparin moiety is a heparin fragment. Heparin fragments can be produced using techniques well known in the art. Suitably, the fragment is a fragment of native heparin produced by a method comprising degrading (e.g. fragmenting) native heparin. As exemplified in example 2e below, heparin fragments may be prepared by partial nitrous acid cleavage of native heparin, optionally followed by fractionation by gel chromatography. Alternatively, heparin fragments may be produced synthetically. The synthetic production may include chemical enzymatic and/or conventional organic chemical methods.

The anticoagulant activity of heparin is primarily dependent on the pentasaccharide sequence that binds Antithrombin (AT) ('active pentasaccharide sequence' or 'active sequence'; see figure 23). Suitably, the heparin fragment comprises an active pentasaccharide sequence.

For example, heparin fragments can have a length of 5-30, such as 5-20, such as 5-18, such as 5-17, such as 5-10, such as 6-10 sugar residues. Alternatively, for example, heparin fragments can have a length of 6-30, such as 6-20, such as 6-18, such as 6-17, sugar residues.

US6,461,665B1 (Scholander; incorporated herein by reference) discloses improving the antithrombotic activity of surface-immobilized heparin by treating the heparin prior to immobilization. This improvement is achieved by treating heparin at elevated temperature or at elevated pH, or by contacting heparin with a nucleophilic catalyst, such as an amine, alcohol or thiol, or a fixed amino, hydroxyl or thiol group.

The anticoagulant substance is covalently immobilized on the surface of the solid object and therefore does not substantially elute or leach from the solid object. As described below, the anticoagulant substance may be covalently immobilized by different methods.

The anticoagulant substance is covalently linked to the outermost layer of the cationic polymer.

Suitably, the anticoagulant substance is attached to the end of the cationic polymer, especially when the anticoagulant substance is a heparin moiety. Thus, in one embodiment, the anticoagulant substance is a terminally linked anticoagulant moiety. In a particular embodiment, the anticoagulant substance is a terminally linked heparin moiety. Where applicable, the anticoagulant substance is preferably linked by its reducing end. Thus, in one embodiment, the anticoagulant substance is linked through its reducing end. In a particular embodiment, the anticoagulant substance is a terminally linked heparin moiety linked through its reducing end (sometimes referred to as position C1 of the reducing end). The advantage of end-linking, in particular reducing end-linking, is that the biological activity of the anticoagulant substance (e.g. heparin fraction) is maximized compared to the linking of the anticoagulant substance (e.g. heparin fraction) at other positions due to the enhanced availability.

Representative terminal attachment methods are described in EP 008686B 1 (Larm; which is incorporated herein by reference in its entirety), which discloses a method of covalently binding oligomeric or polymeric organic materials to different types of substrates comprising primary amino groups. The substance to be coupled, which may be heparin, is degraded by diazotisation to form fragments of the substance with free terminal aldehyde groups. The material fragment then reacts with the amino group of the substrate via its aldehyde group to form a schiff base, which is then converted (by reduction) to a secondary amine. The advantage of heparin end ligation, in particular of reduced end ligation (as described above in EP 008686B 1), is that the biological activity of the heparin moiety is maximized due to the higher availability of antithrombin interaction sites than other ligation in the heparin moiety.

The anticoagulant substance may be covalently linked to the outermost layer of the cationic polymer through a linking group. Thus, in one embodiment, the anticoagulant substance is covalently linked through a linking group.

In one embodiment, the linking group comprises a secondary amine. Representative methods for covalently attaching heparin moieties to polymers via secondary amines are described in EP 008686B 1.

In one embodiment, the linking group comprises a secondary amine. Representative methods of covalently bonding heparin moieties to polymers by amidation reactions involving N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) or 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) are described in WO2012/123384A1 (which is incorporated herein by reference in its entirety).

In one embodiment, the linking group comprises 1,2, 3-triazole. Representative methods of covalently attaching heparin moieties to polymers by 1,2, 3-triazole linkages are described in WO2010/029189A2 (carboda AB, which is incorporated herein by reference in its entirety). This document describes azide-or alkyne-functionalization of polyamines; preparation of alkyne-and azide-functionalized heparin (natural and nitrous acid degraded heparin); and reacting the derivatized heparin with the derivatized polymer via a1, 2, 3-triazole linking group.

In one embodiment, the linking group comprises a thioether. Representative methods of covalently attaching heparin moieties to polymers by thioether linkages are described in WO2011/110684A1 (carboda AB et al, incorporated herein by reference in its entirety).

Cationic polymers

The cationic polymer may be a linear polymer, but is more typically a branched polymer, such as a hyperbranched polymer. In one embodiment, the cationic polymer is a branched cationic polymer. The cationic polymer is optionally crosslinked. In one embodiment, the cationic polymer comprises primary/secondary amine groups. In one embodiment, the cationic polymer is a polyamine, which is optionally crosslinked. Suitably, the cationic polymer (e.g. polyamine) has a molecular weight of 5kDa to 3,000kDa, such as 5kDa to 2,000kDa,5kDa to 1,500kDa,5kDa to 1,000kDa,5kDa to 800kDa,5kDa to 500kDa,5kDa to 300kDa or 5kDa to 200kDa or 800kDa to 3,000kDa. When the cationic polymer (e.g., polyamine) is crosslinked, it is suitably crosslinked using an aldehyde crosslinking agent such as crotonaldehyde and/or glutaraldehyde. In one embodiment, the cationic polymer is a polyalkyleneimine, such as a polyethyleneimine.

The cationic polymer forms part of a layer-by-layer coating of cationic polymer and anionic polymer formed by alternately treating the surface of the solid object with layers of cationic and anionic polymer. Bilayer is defined herein as a layer of a cationic polymer and an anionic polymer. In layer-by-layer coatings, the cationic polymer is usually applied before the anionic polymer, i.e. the surface of the solid object is usually first treated with a first layer of cationic polymer according to claim 1) (step i), followed by the application of the first layer of anionic polymer in step ii according to claim 1). Depending on the number of double layers required, further layers of cationic and anionic polymers may be applied (step iii in claim 1). When the final (and possibly also the first) bilayer of cationic and anionic polymer is completed, a layer of cationic polymer is then applied (corresponding to step iv in claim 1). The layer of cationic polymer (i.e., the outermost layer) is then treated with an anticoagulant substance to covalently attach the anticoagulant substance to the cationic polymer layer. Thus, the outer coating of the cationic polymer may be considered to "comprise" the anticoagulant substance. Of the layer-by-layer coatings, the innermost layer is a layer of cationic polymer and the outermost layer is an outer coating of cationic polymer to which the anticoagulant substance is covalently linked (see fig. 1).

In one embodiment, the cationic polymer of step i) is a polyamine, which is optionally crosslinked. In one embodiment, the cationic polymer of step iv) is a polyamine, which is optionally crosslinked. In one embodiment, the cationic polymer of step i) is the same as the cationic polymer of step iv).

WO2012/123384A1 (Gore Enterprise Holdings, inc et al, incorporated herein by reference in its entirety) discloses a device having a coating comprising a plurality of hyperbranched polymer molecules with an anticoagulant substance, in particular heparin. Such hyperbranched polymer molecules can be used for the outermost layer of the cationic polymer, i.e. such hyperbranched polymer can be used as cationic polymer of step iv) and then modified in step v) to carry an anticoagulant substance.

Anionic polymers

Anionic polymers suitable for the present invention carry moieties derived from-COOH, -SO ₃ H and-PO ₃ H ₂ Is a deprotonated functional group of (a). Thus, in one embodiment, the anionic polymer is a polymer comprising a polymer selected from the group consisting of-CO ₂ ^- 、-SO ₃ ^- 、-PO ₃ H ^- and-PO ₃ ^2- Polymers of groups. Suitably, the anionic polymer is a polymer comprising-SO ₃ ^- Polymers of groups. More suitably, the deprotonated functional groups carried by the anionic polymer are formed from-SO ₃ ^- And (3) groups.

The anionic polymer is suitably an anionic glycosaminoglycan or polysaccharide. The anionic nature of the polymer typically derives from carboxylate or sulfate groups along the polymer chain. Thus, in one embodiment, the anionic polymer is a glycosaminoglycan or polysaccharide bearing carboxylate and/or sulfate groups, in particular a glycosaminoglycan bearing carboxylate and/or sulfate groups. The anionic polymer may be branched or unbranched. In one embodiment, the anionic polymer and the anticoagulant substance are not the same.

In one embodiment, the anionic polymer is optionally crosslinked.

In one embodiment, the anionic polymer is selected from dextran sulfate, hyaluronic acid, poly (2-acrylamido-2-methyl-1-propanesulfonic acid), poly (2-acrylamido-2-methyl-1-propanesulfonic acid-co-acrylonitrile) acrylonitrile, poly (acrylic acid), polyanisole sulfonic acid, poly (sodium 4-styrenesulfonate), poly (4-styrenesulfonic acid-co-maleic acid), poly (vinyl sulfate), polyethylene sulfonic acid, and salts thereof. Suitably, the anionic polymer is dextran sulphate.

Dextran sulfate is a sulfated polymer of anhydroglucose. The degree of sulfation of dextran sulfate and thus the sulfur content can vary.

In some embodiments the sulfur content is 10% to 25% by weight, for example 15% to 20% by weight.

In one embodiment, the anionic polymer is characterized as having a total molecular weight of 750kDa to 10,000kDa, e.g., 1,000kDa to 10,000 kDa. In one embodiment, the anionic polymer is characterized as having a total molecular weight of 650kDa to 1,000kDa, e.g., 750kDa to 1,000 kDa. In one embodiment, the anionic polymer is characterized as having a total molecular weight of 1,000kDa to 4,500kDa, such as 2,000kDa to 4,500 kDa. In one embodiment, the anionic polymer is characterized as having a total molecular weight of 4,500kDa to 7,000 kDa. In one embodiment, the anionic polymer is characterized as having a total molecular weight of 7,000kDa to 10,000 kDa. Suitably, the total molecular weight of the anionic polymer is determined according to evaluation method G.

In one embodiment, the anionic polymer is characterized by having a solution charge density of between >4 to 7 μeq/g, for example between >5 to 7 μeq/g. Suitably, the solution charge density of the anionic polymer is determined according to evaluation method H.

Coated bilayers of cationic and anionic polymers

The method of the present invention involves forming a solid object having a surface comprising a layered coating of cationic and anionic polymers. As described above, a bilayer is defined herein as a layer of a cationic polymer and an anionic polymer (see fig. 1).

Layered coatings comprise one or more coating bilayers, for example 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more coating bilayers. When more than one coating bilayer is applied, steps i) and ii) are repeated, i.e. step iii) is not optional. In one embodiment of the process of the invention, step iii) is not optional. In this embodiment, step iii) is repeated a number of times, for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 times, necessary to achieve the desired coating bilayer. In one embodiment of the method of the invention, in step iii), steps i) and ii) are repeated 1 to 10 times, for example 1, 2, 3, 4, 5 or 6 times.

If step iii) is not optional (i.e. when steps i) and ii) are repeated one or more times), the exact process conditions for each repetition need not be the same (e.g. the salt form and/or concentration used in treating the surface with the anionic polymer in step ii) need not be the same in each repetition). In one embodiment, the processing conditions (e.g., the type and/or concentration of salt used in treating the surface with the anionic polymer in step ii) are the same in each repetition.

Method steps

The present invention provides a method for preparing a solid object having a surface comprising a layered coating of cationic and anionic polymers, wherein the outer coating comprises an anticoagulant substance, the method comprising the steps of:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

It should be noted that steps i) -v) are performed sequentially in the given order, i.e. each step of steps i) -iv) is implicitly followed by "then". This does not exclude that one or more further steps are carried out between each of the specified steps i) -v). Thus, in one embodiment, the method of the invention further comprises steps between step i) and step ii), between step ii) and step iii), between step iii) and step iv) or between step iv) and step v).

For example, it should be understood that the washing step may be performed between the processing steps described.

The inventors have surprisingly found that the salt concentration of step ii), i.e. the salt concentration present when applying the anionic polymer coating, influences the resulting properties of the coating of the solid object, in particular the antithrombotic properties of the final solid object. The inventors have found that when step ii) is carried out at a salt concentration of 0.25M-5.0M, the properties of the coating of the resulting solid object, in particular the antithrombotic properties of the final solid object, can be improved, as shown in examples 2a and 3 a.

In one embodiment, step ii) is carried out at a salt concentration of 0.25M-4.0M, such as 0.25M-3.0M, 0.5M-3.0M, 1.0M-3.0M, 1.5M-3.0M, 0.25M-1.5M, 0.5M-1.5M, 0.75M-1.5M or 1.0M-2.0M, especially a salt concentration of 1.0M-3.0M, such as 1.0M-2.0M or 0.75M-1.5M or 1.5M-3.0M.

In one embodiment, the salt is an inorganic salt. Suitably, the salt is selected from the group consisting of sodium, potassium, magnesium, calcium, lithium, ammonium, barium and strontium salts.

In one embodiment, the salt is an inorganic sodium salt.

In one embodiment, the salt is selected from sodium chloride, sodium sulfate, sodium hydrogen phosphate, and sodium phosphate.

In one embodiment, the salt is sodium chloride.

In one embodiment, the salt is not sodium chloride.

In one embodiment, the salt is sodium chloride at a concentration of 0.25M to 3.0M, such as 0.5M to 3.0M, such as 1.0M to 3.0M, such as 1.5M to 3.0M.

In one embodiment, the salt is sodium sulfate at a concentration of 0.25M to 1.5M, such as 0.5M to 1.5M, such as 0.75M to 1.5M.

In one embodiment, the salt is sodium hydrogen phosphate at a concentration of 0.25M to 3.0M, such as 0.5M to 3.0M, such as 1.0M to 2.0M.

In one embodiment, the salt is sodium phosphate at a concentration of 0.25M to 3.0M, such as 0.5M to 3.0M, such as 1.0M to 2.0M.

The surface of the solid object may optionally be subjected to a pretreatment step prior to step i) (treating the surface of the solid object with the cationic polymer).

The pretreatment step may be a cleaning step to improve adhesion and surface coverage of the subsequent coating. Suitable cleaning agents include solvents such as alcohols, solutions of high pH, for example solutions comprising a mixture of an alcohol and an aqueous solution of a hydroxide compound (e.g. sodium hydroxide), sodium hydroxide solutions, solutions comprising tetramethylammonium hydroxide (TMAH), acidic solutions such as piranha solutions (a mixture of sulfuric acid and hydrogen peroxide), alkaline piranha solutions, and other oxidizing agents including combinations of sulfuric acid and potassium permanganate or different types of peroxosulfuric acid or peroxodisulfuric acid solutions (which may also be ammonium, sodium and potassium salts), or by subjecting the solid object to a plasma treatment in an air, argon or nitrogen atmosphere or combinations thereof.

Thus, in one embodiment, the method of the invention further comprises a pretreatment step prior to step i). Suitably, the pre-treatment step is a cleaning step.

Alternatively, the pretreatment step may comprise, prior to the coating steps i) -v), covering the surface of the solid object to be coated according to steps i) -v) with a material such as a polymer or a primer. The "preparatory" coating may, for example, allow the surface of the solid object to be coated to be "engraved" or modified to produce a desired surface topography or texture, thereby optimizing the subsequent layered coating process. The additional coating may also improve the adhesion of the subsequent layered coating, in particular helping to maintain its integrity during processing. An example of such a primer layer on a solid object is a coating applied using Chemical Vapor Deposition (CVD). Another example of such a primer layer on a solid object is a coating of polydopamine or an analogue thereof.

In one embodiment, the pretreatment step comprises treating the surface of the solid object with a polymer selected from the group consisting of polyolefin, polyisobutylene, ethylene-alpha-olefin copolymer, acrylic polymer, acrylic copolymer, polyvinyl chloride, polyvinylmethyl ether, polyvinylidene fluoride, polyvinylidene chloride, fluoropolymers (e.g., expanded polytetrafluoroethylene (ePTFE), polytetrafluoroethylene (PTFE), fluorinated ethylene-propylene (FEP), perfluorocarbon copolymers such as tetrafluoroethylene perfluoroalkyl vinyl ether (TFE/PAVE) copolymers, copolymers of Tetrafluoroethylene (TFE) with perfluoromethyl vinyl ether (PMVE), copolymers of TFE with functional monomers comprising acetate, alcohol, amine, amide, sulfonate functionalities, and the like, as described in U.S. patent nos. 8,658,707 (Gore and associas), which are incorporated herein in their entirety; and combinations thereof), polyacrylonitrile, polyvinyl ketone, polystyrene, polyvinyl acetate, ethylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins, nylon 12, block copolymers of nylon 12, polycaprolactone, polyoxymethylene, polyethers, epoxy resins, polyurethanes, rayon-triacetate, cellulose acetate, cellulose butyrate, celluloid, nitrocellulose, cellulose propionate, cellulose ethers, carboxymethyl cellulose, chitin, polylactic acid, polyglycolic acid, polylactic acid-polyethylene oxide copolymers, polyethylene glycols, polypropylene glycols, polyvinyl alcohols, elastomeric polymers, such as silicones (e.g., polysiloxanes or substituted polysiloxanes), polyurethanes, thermoplastic elastomers, ethylene vinyl acetate copolymers, polyolefin elastomers, EPDM rubbers, and mixtures thereof.

In one embodiment, a method for manufacturing a solid object as described herein is provided, which method consists of steps i) -v) as defined herein, i.e. the solid object is free of other coatings than those produced by steps i) -v).

The solid objects coated according to the method of the invention may be sterilized. Suitable sterilization methods include, but are not limited to, sterilization using ethylene oxide, vapor hydrogen peroxide, plasma phase hydrogen peroxide, dry heat, autoclave steam sterilization, chlorine dioxide sterilization, gamma ray sterilization, or electron beam sterilization.

As shown in example 7, a solid object coated according to the method of the present invention was subjected to elevated temperature and humidity and maintained its antithrombotic properties. Conditions of elevated temperature and humidity can be used as a simulation of stringent sterilization conditions, particularly ethylene oxide sterilization conditions. Thus, it is expected that solid objects coated according to the method of the present invention may be stably sterilized.

Coating characteristics

Typically, the coating has an average total thickness of about 10nm to about 1000nm, such as about 10nm to about 800nm, such as about 10nm to about 500nm, about 10nm to about 400nm, about 10nm to about 300nm, about 10nm to about 200nm, or about 10nm to about 100 nm. The coating thickness can be measured using a suitable coating thickness analyzer or gauge, by using X-ray photoelectron spectroscopy with depth measurement spectra (see evaluation method J) or by using a quartz crystal microbalance with dissipation (see evaluation method O). Suitably, the coating thickness is measured using evaluation method O.

In one embodiment, a solid object coated according to the method of the invention has at least 1pmol/cm ² Surfaces for binding ATIII, e.g. at least 2pmol/cm ² Surface for binding ATIII, at least 3pmol/cm ² Surface for binding ATIII, at least 4pmol/cm ² For binding to surfaces of ATIII or at least 5pmol/cm ² The activity of the anticoagulant substance (in particular heparin activity) for binding to the surface of ATIII is suitably determined according to evaluation method B.

In one embodiment, the antithrombotic surface of the solid body has at least 1pmol/cm ² Surfaces for binding ATIII, e.g. at least 2pmol/cm ² Surface for binding ATIII, at least 3pmol/cm ² Surface for binding ATIII, at least 4pmol/cm ² For binding to surfaces of ATIII or at least 5pmol/cm ² The activity of the anticoagulant substance (in particular heparin activity) for binding to the surface of ATIII is suitably determined according to evaluation method B.

In one embodiment, the solid object coated according to the method of the invention has at least 5pmol/cm ² Surfaces for binding HCII, e.g. at least 12pmol/cm ² Surface for binding HCII, at least 20pmol/cm ² Surface for binding HCII, at least 50pmol/cm ² The activity of the anticoagulant substance (in particular heparin activity) for binding to the surface of HCII is suitably determined according to evaluation method M.

In one embodiment, the antithrombotic surface of the solid body has at least 5pmol/cm ² Surfaces for binding HCII, e.g. at least 12pmol/cm ² Surface for binding HCII, at least 20pmol/cm ² Surface for binding HCII, at least 50pmol/cm ² For knotsThe surface anticoagulant activity (in particular heparin activity) of HCII is suitably determined according to evaluation method M.

In one embodiment, the solid object coated according to the method of the invention has a blood contacting property of at least 80% preserved platelets, e.g. at least 85% preserved platelets, e.g. at least 90% preserved platelets, suitably determined according to evaluation method E.

In one embodiment, the antithrombotic surface of the solid object has a blood contacting property of at least 80% preserved platelets, such as at least 85% preserved platelets, such as at least 90% preserved platelets, suitably determined according to evaluation method E.

In one embodiment, the solid object coated according to the method of the invention has a f1+2 value of <10,000pmol/L, e.g. less than 7,500pmol/L, less than 5,000pmol/L or less than 4,000pmol/L, suitably it is determined according to evaluation method F.

In one embodiment, the antithrombotic surface of the solid object has a f1+2 value of <10,000pmol/L, less than 7,500pmol/L, less than 5,000pmol/L or less than 4,000pmol/L, suitably determined according to evaluation method F.

In one embodiment, the anticoagulant substance is a heparin fraction, wherein the solid body has at least 1 μg/cm ² For example at least 2. Mu.g/cm ² At least 4. Mu.g/cm ² At least 5 μg/cm ² Or at least 6. Mu.g/cm ² Heparin concentration of (c). Suitably, it is determined according to evaluation method a.

Suitably, the zeta potential profile of a solid object coated according to the method of the invention may be determined using evaluation method D. FIG. 22 shows a typical zeta potential spectrum of a solid object coated according to the method of the invention, indicating that a zeta potential spectrum, in particular the isoelectric point (IEP) (1A), corresponding to a specific pH value at a zeta potential of 0mV, can be qualitatively and definable using a variety of parameters; the overall minimum (overall minimum) of the curve corresponds to the pH (2A) at which the zeta potential (2B) is minimal; and a delta value (delta) corresponding to the difference between the zeta potential at the overall minimum (3A) and the zeta potential at pH 9 (3B).

As can be seen in fig. 7-13 (examples 4a and 4 b), similar zeta potential spectra have been obtained for solid objects coated with dextran sulfate 4-7 according to the method of the present invention. Thus, in at least its preferred aspect, the zeta potential spectrum can be regarded as a potential fingerprint of a solid object coated according to the method of the invention. From this potential fingerprint, it is preferred that IEP (1A) is below pH 3, the overall minimum (2A) of the curve is below pH 5, and the delta value, i.e. the difference between the zeta potential at overall minimum (3A) and the zeta potential at pH 9 (3B), is at least 20mV. Suitably, these parameters are determined according to evaluation method D.

Suitably, the solid object coated according to the method of the invention has a zeta potential spectrum with an isoelectric point (IEP) below pH 3, as the acidic properties of heparin predominate on the coated surface. In contrast, inert polymeric materials have IEPs at about pH 4. The swelling properties of the sample with acidic groups towards the alkaline region were observed. This swelling will force the shear plane towards the bulk and should result in a lower absolute zeta potential value (near 0 mV). For example, a high delta value of zeta potential correlates with high antithrombotic properties when evaluated according to evaluation methods B, M, E or F. In addition, the higher salt concentration used in the method of the invention gives a lower absolute zeta potential value in the alkaline region, which is again related to the antithrombin binding value, no matter what the salt tested is. Without being limited by theory, this may be explained by the increased ingress of antithrombin to heparin molecules in the coating which may undergo swelling.

Therapeutic method

The solid objects, in particular medical devices, coated according to the method of the invention as described above have utility in medical treatment.

In one aspect of the present invention, there is provided a solid object (in particular a medical device) coated according to the method of the invention described above for use in the treatment of tissue in the human or animal body. The tissue to be treated includes any body cavity, space or hollow organ passage, such as blood vessels, urinary tracts, intestinal tracts, nasal cavities, nerve sheaths, intervertebral regions, bone cavities, esophagus, uterine cavities, pancreatic ducts and bile ducts, rectum, and body spaces where vascular grafts, stents, prostheses or other types of medical implants have been previously implanted. In another aspect of the invention, a solid object (e.g., a medical device) coated according to the methods of the invention described above can be deployed to treat an arterioma in the brain.

A coated solid object as described herein (in particular a medical device) may have the following uses: for removing obstructions such as emboli and thrombi from blood vessels, as an expansion device for restoring patency to an occluded body passageway, as an occlusion device for selectively delivering a tool for occluding or filling a passageway or space, and as a centering mechanism for surgical instruments such as catheters within lumens.

In one embodiment there is provided a solid object (in particular a medical device, such as a stent, graft or stent-graft) coated according to the method of the invention described above for use in the prevention or treatment of stenosis or restenosis in a human blood vessel. In another embodiment, a solid object (in particular a medical device, such as a stent, graft or stent-graft) coated according to the method of the invention as described above is provided for the prevention or treatment of vascular stenosis or restenosis, wherein a previously placed eluting structure fails. In another embodiment, solid objects (particularly medical devices, such as stents, grafts or stent-grafts) coated according to the methods of the invention described above may be used to establish or maintain arteriovenous access sites, such as those used during renal dialysis. In another embodiment, a solid object (in particular a medical device, such as a stent, graft or stent-graft, such as a vascular graft) coated according to the method of the invention described above may be used to redirect fluid around an occlusion or a vascular stenosis. In another embodiment, a solid object (particularly a medical device such as a stent, graft or stent-graft) coated according to the methods of the invention described above may be deployed to restore patency to the diseased vascular area or to exclude an aneurysm. In yet another embodiment, a solid object (particularly a medical device such as a stent, graft or stent-graft) coated according to the methods of the invention described above may be deployed to strengthen a diseased vessel after angioplasty. In yet another embodiment, a solid object (in particular a medical device, such as a stent, graft or stent-graft) coated according to the method of the invention described above may be deployed in the brain using balloon-assisted or coil-assisted methods.

In one embodiment, a solid object, in particular a medical device, coated according to the method of the invention described above may be used for Percutaneous Transluminal Angioplasty (PTA) of patients suffering from peripheral arterial occlusive disease.

Another aspect of the invention provides a method of preventing or treating stenosis or restenosis, the method comprising implanting a solid object, in particular a medical device, coated according to the method of the invention as described above, into said blood vessel of a human body.

Other embodiments of the invention

The embodiments and preferred embodiments described above in relation to the process of the present invention apply equally to the following embodiments.

In one embodiment, a method is provided for preparing a solid object having a surface comprising a layered coating of cationic and anionic polymers, wherein the overcoat is a layer comprising cationic polymers, the method comprising the steps of:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times; and

iv) treating the surface with a cationic polymer;

And wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

In one embodiment, a solid object is provided having a surface comprising a layered coating of cationic and anionic polymers, wherein the outermost layer is a layer comprising cationic polymer;

and wherein the anionic polymer is characterized by having: (a) a total molecular weight of 650kDa to 10,000 kDa; and (b) a solution charge density of > 4. Mu. Eq/g.

In one embodiment, a method is provided for preparing a solid object having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating is a layer comprising an anionic polymer, the method comprising the steps of:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

In one embodiment, a solid object is provided having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating is a layer comprising an anionic polymer; and wherein the anionic polymer is characterized by having: (a) a total molecular weight of 650kDa to 10,000 kDa; and (b) a solution charge density of > 4. Mu. Eq/g.

In one embodiment, a method is provided for preparing a solid object having a surface comprising a layered coating of cationic and anionic polymers, wherein the outer coating comprises an anticoagulant substance, the method comprising the steps of:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

In one embodiment, a solid object is provided having a surface comprising a layered coating of cationic and anionic polymers, wherein the outer coating is a layer comprising a cationic polymer covalently linked to an anticoagulant substance; and wherein the anionic polymer is characterized by having: (a) a total molecular weight of 650kDa to 10,000 kDa; and (b) a solution charge density of > 4. Mu. Eq/g. Suitably, the anionic polymer is applied to the surface at a salt concentration of from 0.25M to 5.0M, for example from 0.25M to 4.0M or from 0.25M to 3.0M.

In one embodiment, a solid object is provided having a surface comprising a layered coating of cationic and anionic polymers, wherein the outer coating is a layer comprising a cationic polymer covalently linked to an anticoagulant substance; and wherein the anionic polymer is characterized by having: (a) a total molecular weight of 650kDa to 1,000 kDa; and (b) a solution charge density of > 4. Mu. Eq/g. Suitably, the anionic polymer is applied to the surface at a salt concentration of from 0.25M to 5.0M, for example from 0.25M to 4.0M or from 0.25M to 3.0M.

In one embodiment, a solid object is provided having a surface comprising a layered coating of cationic and anionic polymers, wherein the outer coating is a layer comprising a cationic polymer covalently linked to an anticoagulant substance; and wherein the anionic polymer is characterized by having: (a) a total molecular weight of 1,000kDa to 4,500 kDa; and (b) a solution charge density of > 4. Mu. Eq/g. Suitably, the anionic polymer is applied to the surface at a salt concentration of from 0.25M to 5.0M, for example from 0.25M to 4.0M or from 0.25M to 3.0M.

In one embodiment, a solid object is provided having a surface comprising a layered coating of cationic and anionic polymers, wherein the outer coating is a layer comprising a cationic polymer covalently linked to an anticoagulant substance; and wherein the anionic polymer is characterized by having: (a) a total molecular weight of 4,500kDa to 7,000 kDa; and (b) a solution charge density of > 4. Mu. Eq/g. Suitably, the anionic polymer is applied to the surface at a salt concentration of from 0.25M to 5.0M, for example from 0.25M to 4.0M or from 0.25M to 3.0M.

In one embodiment, a solid object is provided having a surface comprising a layered coating of cationic and anionic polymers, wherein the outer coating is a layer comprising a cationic polymer covalently linked to an anticoagulant substance; and wherein the anionic polymer is characterized by having: (a) a total molecular weight of 7,000kDa to 10,000 kDa; and (b) a solution charge density of > 4. Mu. Eq/g. Suitably, the anionic polymer is applied to the surface at a salt concentration of from 0.25M to 5.0M, for example from 0.25M to 4.0M or from 0.25M to 3.0M.

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

the anionic polymer is dextran sulfate;

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

In one embodiment, a solid object is provided having a surface comprising a layered coating of cationic and anionic polymers, wherein the outer coating is a layer comprising a cationic polymer covalently linked to an anticoagulant substance; the anionic polymer is a polymer comprising-SO ₃ ^- A polymer of groups, and wherein the anionic polymer is characterized as having: (a) a total molecular weight of 650kDa to 10,000 kDa; and (b) a sulfur content of 10% to 25% by weight of the anionic polymer.

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times; and

iv) treating the surface with a cationic polymer;

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times; and

iv) treating the surface with a cationic polymer;

the anionic polymer is dextran sulfate;

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

In one embodiment, a solid object is provided having a surface comprising a layered coating of cationic and anionic polymers, wherein the outermost layer is a layer comprising cationic polymer; the anionic polymer is a polymer comprising-SO ₃ ^- A polymer of groups, and wherein the anionic polymer is characterized as having: (a) a total molecular weight of 650kDa to 10,000 kDa; and (b) a sulfur content of 10% to 25% by weight of the anionic polymer.

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

the anionic polymer is dextran sulfate;

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

In one embodiment, a solid object is provided having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating is a layer comprising an anionic polymer; anions (v-v) The polymer is a polymer comprising-SO ₃ ^- A polymer of groups, and wherein the anionic polymer is characterized as having: (a) a total molecular weight of 650kDa to 10,000 kDa; and (b) a sulfur content of 10% to 25% by weight of the anionic polymer.

Items of the invention

Other items of the invention:

1. a method for preparing a solid object having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating comprises an anticoagulant substance, the method comprising the steps of:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

2. The method for producing a solid object of item 1, wherein the anionic polymer is dextran sulfate.

3. The method for producing a solid object of item 1 or item 2, wherein the anionic polymer is characterized by having a total molecular weight of 750kDa to 10,000kDa, for example 1,000kDa to 10,000kDa.

4. The method for producing a solid object according to any one of items 1 to 3, wherein the anionic polymer is characterized by having a solution charge density of from >4 to 7 μeq/g, for example from >5 to 7 μeq/g.

5. The method for producing a solid object according to any one of items 1 to 4, wherein step ii) is performed at a salt concentration of 0.25M to 4.0M, for example 0.25M to 3.0M.

6. The method for producing a solid object according to any one of items 1 to 5, wherein the salt is selected from sodium chloride, sodium sulfate, sodium hydrogen phosphate and sodium phosphate, and particularly sodium chloride.

7. The process for preparing a solid object according to any one of items 1 to 6, wherein the cationic polymer of step i) is a polyamine, which is optionally crosslinked; and/or

Wherein the cationic polymer of step iv) is a polyamine, which is optionally crosslinked.

8. The method for producing a solid object according to any one of items 1 to 7, wherein the anticoagulant substance is a heparin moiety, such as a terminally linked heparin moiety, which is terminally linked by its reducing end.

9. The method for producing a solid object according to any one of items 1 to 8, wherein the solid object is an antithrombotic medical device.

10. A solid object having a surface comprising a layered coating of cationic and anionic polymers, wherein the outer coating is a layer comprising a cationic polymer covalently linked to an anticoagulant substance;

11. The solid object of item 10, wherein the anionic polymer is characterized by having a total molecular weight of 650kDa-1,000kDa or 1,000kDa-4,500kDa or 4,500kDa-7,000kDa or 7,000kDa-10,000 kDa.

12. The solid object of any of clauses 10 or 11, wherein the anionic polymer is applied to the surface at a salt concentration of 0.25M-5.0M, such as 0.25M-4.0M or 0.25M-3.0M.

13. A method for preparing a solid object having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating is a layer comprising a cationic polymer, the method comprising the steps of:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times; and

iv) treating the surface with a cationic polymer;

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

14. A solid object having a surface comprising a layered coating of cationic and anionic polymers, wherein the outermost layer is a layer comprising cationic polymer;

15. A method for preparing a solid object having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating is a layer comprising an anionic polymer, the method comprising the steps of:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

the anionic polymer is characterized by having: (a) a total molecular weight of 650kDa 10,000 kDa; and (b) a solution charge density of >4 μeq/g;

And wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

Advantages are that

In at least some embodiments, solid objects coated according to the methods of the present invention are expected to have one or more of the following advantages or benefits:

for example, as determined using evaluation method C (toluidine blue staining test) or evaluation method I (SEM), it is possible to obtain a coating of anticoagulant substance with a uniform distribution and relatively smooth;

a uniform coating can be obtained which will mask the inherent properties of the solid object, for example for improving the antithrombotic properties of the device, irrespective of the material from which it is made;

for example, as determined by using evaluation method B or M, a coating having good anticoagulant activity, e.g. heparin activity, can be obtained;

due to their covalent bonding, antithrombotic coatings are obtained which do not exude anticoagulant substances such as heparin, whereby a long lifetime can be obtained;

coatings can be obtained that retain their properties when sterilized (e.g. with EO);

self-healing coatings can be obtained due to the possibility of reversible ionic interactions between the layers;

for example, a coating with good biocompatibility can be obtained, as determined using evaluation method N;

A coating is available which reduces the need for systemic administration of an anticoagulant such as heparin and reduces the possibility of contact activation, for example as determined using evaluation method E (platelets) and/or evaluation method F (blood circulation);

solid objects having a combination of anti-inflammatory and anti-thrombotic properties as determined by using evaluation method N may be obtained, which may be beneficial in certain applications, such as cardiovascular applications;

an analysis or separation device with good binding capacity for biomolecules can be obtained; and

an analysis or separation device with a long lifetime of heparin activity can be obtained.

The invention covers all combinations of the illustrated groups and embodiments of the above groups.

Abbreviations (abbreviations)

Ac acetyl group

ABS acrylonitrile butadiene styrene

ATIII antithrombin III

CNS central nervous system

CPB cardiopulmonary bypass

CVC central venous catheter

CVD chemical vapor deposition

Da daltons

DI deionized water

EDC 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide

EO ethylene oxide

EPDM ethylene propylene diene monomer (M-type)

ePTFE expanded polytetrafluoroethylene

FEP fluorinated ethylene-propylene

GPC gel permeation chromatography

HCII heparin cofactor II

HIT heparin-induced thrombocytopenia

Isoelectric point of IEP

M molar concentration

MBTH 3-methyl-2-benzothiazolinone hydrazone hydrochloride

PAVE perfluoroalkyl vinyl ethers

PES-Na polyethylene sodium sulfate

PTA percutaneous transluminal angioplasty

Central catheter inserted into PIC periphery

PMVE perfluoromethyl vinyl ether

PTFE polytetrafluoroethylene

PUR polyurethanes

PVC polyvinyl chloride

RGD arginyl glycyl aspartic acid

SEM scanning electron microscopy/microscopy

SPDP 3- (2-pyridyldithio) propionic acid N-succinimidyl ester

TFE tetrafluoroethylene

TMAH tetramethyl ammonium hydroxide

TMB 3,3', 5' -tetramethylbenzidine

VA ventricular atrium

VP ventriculo-peritoneal

XPS X-ray photoelectron spectrophotometry

Detailed Description

Examples

General method

Chemical product

Isopropyl alcohol, sodium dihydrogen phosphate dihydrate, sodium sulfate and sodium chloride are available from Sigma Aldrich and VWR Chemicals and are used as received products. Pharmacopoeial quality heparin is treated with nitrous acid and used in the examples essentially as described in EP 008686 A1. Polyamines are available from suppliers as described in US9,101,696B2. Dextran sulfate is available from the suppliers shown in table 1 of example 1. Deionized (DI) water was used in the following examples.

Material

PVC tubing was purchased from Flextubing Products. PUR tubes were purchased from NewAge Industries. Stainless steel sampling tubes were purchased from Helab Mekano AB.

Evaluation method

Parameters evaluated by each method are given in brackets.

Evaluation method A heparin concentration test (quantitative heparin attachment)

Surface immobilized heparin can be quantified by completely degrading heparin and then colorimetrically assaying the reaction products released into solution. Degradation is achieved by reacting the heparin surface with excess sodium nitrite under acidic conditions. Degradation products (mainly disaccharides) were analyzed colorimetrically in reactions with MBTH (3-methyl-2-benzothiazolinone hydrazone hydrochloride) essentially as described in Smith r.l. and Gilkerson E (1979), anal Biochem 98,478-480, incorporated herein by reference in its entirety.

Evaluation method B heparin Activity test (quantitative heparin function Using ATIII)

For the basis ofThe heparin activity of the device can be determined by measuring the capacity or capacity of heparin binding antithrombin III (ATIII) as described in Pasche, et al, "A binding of antithrombin to immobilized heparin under varying flow conditions" (artif. Organics 1991;15:281-491, incorporated herein by reference in its entirety) and Larsen M.L, et al, "Assay of plasma heparin using thrombin and the chromogenic substrate H-D-Phe-Pip-Arg-pNA" (S-2238) (Thromb. Res.1978;13:285-288, incorporated herein by reference in its entirety), and can be used to evaluate antithrombotic properties of the solid object. The washed sample was incubated with excess antithrombin solution to saturate all available antithrombin binding sites on the heparin surface. The nonspecifically adsorbed antithrombin was washed out with saline solution. Subsequently, antithrombin that specifically binds to the surface-bound heparin is released by incubation with high concentration heparin solution. Finally, antithrombin released from the heparin surface was measured in a thrombin inhibition assay based on chromogenic thrombin substrate. The results are expressed as picomoles of antithrombin III (ATIII) per apparent square centimeter of device bound (pmol ATIII/cm) ² Solid object surface). Apparent solid object surface area is not considered for a plurality of covered surfaces nor is the porosity of a solid object composed of a porous material. If the surface of the solid object is porous, the effect of porosity on surface area is not considered in these calculations. For example, for any cylindrical geometry, the apparent surface area of a cylindrical tubular ePTFE vascular graft (made of porous material) with heparin immobilized on a matrix material comprising the inner surface of the tubular graft was calculated as 2ττ rL: wherein r is the inner diameter of the implant; l is the axial length; and pi is digital pi. The method can be used to measure the activity of any anticoagulant substance having ATIII binding activity.

Evaluation method C toluidine blue staining test (heparin distribution)

Heparin distribution was assessed using toluidine blue staining solution. A solution was prepared by dissolving 200mg of toluidine blue in 1L of water. The samples were placed in the staining solution for 2 minutes before extensive water washing was performed. Blue/violet staining indicates that negatively charged heparin molecules are evenly distributed in the outer coating.

Evaluation method D zeta potential measurement value (surface Charge indicator)

The zeta potential of the coating as an indicator of surface charge was determined on a SurPASS instrument. The measurement is performed by circulating an electrolyte, typically a simple electrolyte such as a 1mM solution of KCl or NaCl, over the surface. The resulting streaming potential was measured and used to determine zeta potential. The zeta potential of the coating was measured at a pH in the range of 3 to 9 by adding an acid or base to the solution, respectively. ZETA potential was calculated using equation 1 below as described in t.luxbacher, the ZETA guide, principles of The streaming potential technique, 1 st edition, anton Paar GMBH publication, ISBN 978-3-200-03553-9 (which is incorporated herein by reference in its entirety).

dU/dP = slope of flow potential versus differential pressure

K _B Electrolyte conductivity

η = electrolyte viscosity

Epsilon = dielectric permittivity of electrolyte

ε ₀ =vacuum dielectric constant

Evaluation method E blood circulation evaluation test (measurement of platelet loss)

Blood contact evaluation can be performed on the coated object to evaluate its antithrombotic properties. The method that can be used when the solid object is a tubular device such as a PVC pipe is as follows. First, the lumen side of the coated tube was washed with 0.15M saline at a flow rate of 1mL/min for 15 hours to ensure complete wetting and removal of any loosely bound anticoagulant substance, thereby preserving a stable surface. The washed tubes were then incubated at 20rpm in a Chandler loop model conducted substantially in accordance with Andersson et al (Andersson, j.; sanchez, j.; ekdahl, k.n.; elgue, g.; nilsson, b.; larsson, R.J Biomed Mater Res A2003,67 (2), 458-466, incorporated herein by reference in its entirety). Platelets from fresh blood and blood collected from the circulation are counted in a cell counter to measure the loss of platelets. The massive loss of platelets indicates poor antithrombotic properties of the surface. In contrast, minimal loss of platelets is indicated as antithrombotic surface.

Evaluation method F blood circulation evaluation test (for F1+2 measurement)

The assay of f1+2 (prothrombin fragment) is used as an activation marker for coagulation (i.e. an indirect measurement of thrombin). F1+2 is proportional to thrombin formation and is interpreted as an indirect measure of thrombin generation and can be used to evaluate the antithrombotic properties of solid objects. The quantitative determination of f1+2 in plasma was performed by enzymatic immunoassay using a standard ELISA kit (enzyme-linked immunosorbent assay) (enzygnostf1+2elisa, opbdg03, siemens). The f1+2 antigen in the sample is conjugated to an antibody captured on the coated surface of a 96-well microtiter plate and subsequently detected by peroxidase conjugated anti-f1+2 antibodies. The amount of coupled peroxidase is measured by adding the specific substrate 3,3', 5' -Tetramethylbenzidine (TMB). The enzymatic conversion of the substrate to chromogen is terminated by the addition of dilute sulfuric acid. The absorbance at 450nm in the well is proportional to the concentration of f1+2 in the sample. The concentration of the sample is determined by comparison with a standard curve of known f1+2 concentration.

Evaluation method G molecular weight of anionic Polymer such as dextran sulfate in solution

The molecular weight of the dextran sulfate sample was determined using a Gel Permeation Chromatograph (GPC). Dextran sulfate samples were dissolved in a water-based elution medium and analyzed on a GPC instrument suitable for molecular weights ranging from 1,000Da to 100,000Da (Superose column) or from 100,000Da to 2,000,000Da (Sephacryl column). Dextran sulfate standards of appropriate molecular weight were used to verify the accuracy of the calibration curve. Polymers such as dextran sulfate are dispersed molecules, i.e. having a molecular weight distribution, which can be described by different average molecular weights. The value generally reported is the weight average molecular weight (Mw). See Odian g., principles of Polymerization, 3 rd edition, section 1.4 Molecular weight, p.24 (which is incorporated herein by reference in its entirety), which explains the theory of determining Molecular weight of polymers using GPC techniques. The molecular weight of anionic polymers other than dextran sulfate can also be determined using this method.

Evaluation method H solution Charge Density of anionic Polymer such as dextran sulfate in solution

Quantitative determination of charge density was performed by titration of polyelectrolyte solution (0.001M) (polydiallyl dimethyl ammonium chloride (Poly-Dadmac) and sodium polyethylene sulfate (PES-Na)) using a Mutek particle charge detector. The samples were dissolved in water (maximum viscosity allowed is 6000 mPas) to a concentration of 0.06 g/L. The pH was adjusted to 3 for all sample solutions. 10 mL/sample solution was added for each measurement, and then the appropriate polyelectrolyte solution was titrated at 1 unit intervals every 3 seconds. See Farris et al, charge Density Quantification of Polyelectrolyte Polysaccharides by Conductometric Titration: an Analytical Chemistry Experiment, J.chem.Educ.,2012,89 (1), pp 121-124 (which is incorporated herein by reference in its entirety). The solution charge density of anionic polymers other than dextran sulfate can also be determined using this method.

Evaluation method I scanning electron microscopy with energy dispersive X-ray spectrophotometry (coating coverage) And uniformity of

TM3000 is a tabletop Scanning Electron Microscope (SEM) manufactured by Hitachi, which is used to obtain information about, for example, sample thickness, topography (surface structure), and surface coverage. Higher magnification can be achieved with a desktop SEM compared to a conventional optical microscope, because it is the electrons used to create the image. TM3000 is also equipped with Quantax70. This is an energy dispersive X-ray spectrometer (EDS) for determining the chemical composition of a sample. In addition, there is a rotating/tilting table as an accessory to assist in analyzing the different constituent parts of the sample. The sample was fixed to the rack with a carbon tape (also used as ground) and then placed in the test chamber. The chamber was evacuated to a lower pressure before starting to evaluate the sample. SEM techniques are based on scanning an electron beam across a sample, some of which are reflected back scattered electrons, while others are secondary electrons. The detector is used to measure the current generated by the reflected backscattered electrons. The current is imaged on the display, with each pixel corresponding to the location of the sample. If many electrons are reflected (high electron density), a bright pixel is obtained; if fewer electrons are reflected (low electron density) a darker pixel is obtained.

Evaluation method J X-ray photoelectron spectroscopy with depth measurement Spectrometry (XPS) (coating thickness)

X-ray photoelectron spectroscopy (XPS or ESCA) is the most widely used surface characterization technique that can provide non-destructive chemical analysis of solid materials. The sample is irradiated with unienergy X-rays, causing photoelectrons to be emitted from the top 1nm to 10nm of the sample surface. The electron energy analyzer determines the binding energy of the photoelectrons. All elements except hydrogen and helium can be qualitatively and quantitatively analyzed with a detection limit of about 0.1 to 0.2 atomic percent. The size of the analysis spot ranged from 10 μm to 1.4mm. The surface image of the feature can also be generated using elemental and chemical state mapping. Using angle-dependent measurements, depth-determined spectral analysis can be performed to obtain non-destructive analysis within the top 10nm of the surface, or using destructive analysis (e.g., ion etching) to obtain spectral analysis of the entire coating depth.

Evaluation method K increased temperature and humidity test (general model of Sterilization stability)

The solid objects coated according to the method of the invention are placed in a gas-permeable polyethylene pouch (e.g. a Tyve pouch) and the pouch is placed in a climatic chamber (e.g. Climacell) at 40 ℃ and 50% relative humidity for 1 week, then dried in a vacuum chamber for 2 hours. After this general model was conducted to investigate the sterilization stability, the antithrombotic properties/activation of the coated objects can be evaluated using, for example, evaluation methods E or F.

Evaluation method L stability to ethylene oxide

The solid objects coated according to the method of the present invention were placed in a gas permeable polyethylene pouch (e.g., a Tyve pouch) and subjected to a pretreatment at 50 ℃ and 60% relative humidity for at least 12 hours, followed by exposure to ethylene oxide at 50 ℃ and a pressure of 366mBar for 2 hours. The chamber was then degassed at 50 ℃ for at least 10 hours. Ethylene oxide sterilization can be performed at Synergy Health Ireland ltd. After sterilization, the coated object may be evaluated for antithrombotic properties/activation using, for example, evaluation methods E or F.

Evaluation method M heparin Activity test (quantitative heparin function Using HCII)

For solid objects coated according to the methods of the present invention comprising a heparin coating, the heparin activity of the device can be determined by determining the ability or capacity of heparin to bind heparin cofactor II (HCII) as described in WO2009/064372A2 (Gore Enterprise Holdings, inc.; incorporated herein by reference in its entirety), using the method described in Larsen m.l. et al, in "Assay of plasma heparin using thrombin and the chromogenic substrate H-D-Phe-Pip-Arg-pNA (S-2238)," Thromb Res 13:285-288 (1978) and Pasche b. Et al, in "A binding of antithrombin to immobilized heparin under varying flow conditions." artif. Organics 1991; 15:281-491) and can be used to evaluate the antithrombotic properties of solid objects. The results are expressed as picomolar heparin cofactor Il (HCII) (pmol HCll/cm) bound per apparent square centimeter of solid object surface ² Solid object surface). Apparent solid object surface area is not considered for a plurality of covered surfaces nor is the porosity of a solid object composed of a porous material. If the surface of the solid object is porous, the effect of porosity on surface area is not considered in these calculations. For example, for any cylindrical geometry, the apparent surface area of a cylindrical tubular ePTFE vascular graft (made of porous material) with heparin immobilized on a matrix material comprising the inner surface of the tubular graft was calculated as 2ττ rL: wherein r is the inner diameter of the implant; l is the axial length; and pi is digital pi. The method can be used to measure the activity of any anticoagulant substance having HCII binding activity.

Evaluation methodN-surface biocompatibility

The biocompatibility of the surface of a solid object coated according to the method of the present invention can be assessed as described in Lappegard, K.T 2008, j.biomed.mater.res.87, volumes 129-135, which are incorporated herein by reference in their entirety. Methods that can be used to evaluate inflammatory responses are as follows. First, the coated solid object was washed with 0.15M saline solution for 15 minutes. The wetted coated solid object was placed in heparinized PVC tubing containing whole blood and rotated in the circulation at 20rpm (see Ekdahl k.n., advances in Experimental Medicine and Biology,2013,735,257-2700, incorporated herein by reference in its entirety) as a representative method. After incubation, the blood was centrifuged at 3220g for 15min at 4 ℃. The plasma was then frozen in aliquots at-70 ℃ for subsequent cytokine analysis. Plasma samples were analyzed using a multiplex cytokine assay (Bio-Plex Human Cytokine 27-Plex Panel, bio-Rad Laboratories, hercules, calif.) according to the method described by Lappegard et al (supra).

The negative control was heparinized PVC empty cycles without any device. This represents a non-inflammatory control, whose incubated blood should show no or only a small amount of inflammatory markers. The positive control was a non-heparinized PVC empty cycle without any device. This represents an inflammatory control for which a greater amount of inflammatory markers should be observed. Controls were included to ensure the quality of the experiment and blood.

Evaluation method O-Quartz Crystal microbalance with dissipation (coating thickness)

Q-sense E4 is a crystal microbalance with a dissipative (QCM-D) monitoring instrument. QCM-D is a technique for measuring the mass and structural characteristics of molecular layers and can be regarded as an ultra-sensitive weighing device.

QCM sensors consist of a thin quartz disk, of which the most common is an AT cut crystal. The quartz plate is placed between the two electrodes, and by applying a voltage to the quartz crystal, the quartz plate can be oscillated at its resonance frequency. The change in quartz surface quality induces a change in the frequency of the vibrating crystal involved by the Sauerbrey correlation (see Rodahl, m., et al, quartz crystal microbalance setup for frequency and Q factor measurements in gaseous and liquid environments. Review of scientific environments,1995.66 (7): p.3924-3930 (which is incorporated herein by reference in its entirety.) the coating thickness of a solid object coated according to the method of the present invention is reported as a dry coating thickness.

Evaluation method molecular weight determination of P-heparin fragment component

The molecular weight of the heparin fragment fraction was determined by analytical Gel Permeation Chromatography (GPC) on a system consisting of two Superdex columns (S-75 and S-200) in series, essentially according to the USP <209> low molecular weight heparin molecular weight determination. The peak position was identified based on the elution profile of the "second international standard for molecular weight calibration of low molecular weight heparin" (NIBSC, UK), where the minimum delay peak of this standard is disaccharide.

Evaluation method Q-heparin fragment concentration determination

The amount of isolated heparin fragment in solution was estimated by analysis of uric acid content by carbazole assay in relation to heparin standard curve (Bitter, t.; muir, H.M., anal.Biochem.,1962, (4), 330-334).

Example 1 method of coating solid objects (layered coating of cationic and anionic polymers with an overcoat of anticoagulant substance)

Universal coating method-tubulation

Essentially, cationic and anionic polymers are applied layer by layer to the luminal surface of a length of tubing (e.g., PVC or PUR tubing) using the method described by Larm et al in EP 00867886 A1, EP0495820B1 and EP0086187A1, all incorporated herein by reference in their entirety.

Specifically, the lumen surface of the tube is first cleaned with isopropyl alcohol and an oxidizing agent. The coating bilayer was built up by alternating adsorption of cationic polymer (polyamine, 0.05g/L in water) and anionic polymer (dextran sulfate, 0.1g/L in water). The polyamine is crosslinked with a difunctional aldehyde (crotonaldehyde). Dextran sulfate starting materials were varied as specified in each of the examples below and used in the presence of different sodium salts at different concentrations, as also described in each of the examples below. Each pair of polyamine and sulfated polysaccharide is referred to as a bilayer, i.e., a bilayer is defined as one layer of cationic and anionic polymers, and each bilayer is constructed using the same conditions. The inner lumen surface of the tube is coated with three bilayers (for solid objects coated with a single bilayer, see fig. 1). The final polyamine outermost layer is then adsorbed.

Heparin is then immobilized on the outermost layer of the polyamine by reductive amination substantially as described in Larm et al, EP 008686 A1 and EP0495820B1, both incorporated herein by reference in their entirety.

Universal coating method-steel sampling tube

Any solid object can be coated using the general coating methods described above for pipes. In the following examples using steel coupon, the entire surface of the coupon is coated.

Dextran sulfate used in examples 1.1 to 1.56

Dextran sulfate was evaluated as purchased from different suppliers as shown in table 1.

Table 1-dextran sulfate evaluated in the examples

* Weight average molecular weight (Mw) measured according to evaluation method G

* Solution charge density determined according to evaluation method H

EXAMPLE 1.1 preparation of a coating on PVC pipe Using dextran sulfate 1 and 0.25MNaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 1 was coated at 0.25M NaCl concentration.

EXAMPLE 1.2 preparation of a coating on PVC pipe Using dextran sulfate 1 and 1.7M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 1 was coated at a 1.7M NaCl concentration.

EXAMPLE 1.3 preparation of a coating on PVC pipe Using dextran sulfate 2 and 0.25MNaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 2 was coated at 0.25M NaCl concentration.

EXAMPLE 1.4 preparation of a coating on PVC pipe Using dextran sulfate 3 and 0.05M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 3 was coated at 0.05M NaCl concentration.

EXAMPLE 1.5 preparation of a coating on PVC pipe Using dextran sulfate 3 and 0.1M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 3 was coated at 0.1M NaCl concentration.

EXAMPLE 1.6 preparation of a coating on PVC pipe Using dextran sulfate 3 and 0.25M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 3 was coated at 0.25M NaCl concentration.

EXAMPLE 1.7 preparation of a coating on PVC pipe Using dextran sulfate 3 and 1.0M NaCl concentration

EXAMPLE 1.8 preparation of a coating on PVC pipe Using dextran sulfate 3 and 1.7M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 3 was coated at a 1.7M NaCl concentration.

EXAMPLE 1.9 preparation of a coating on PVC pipe Using dextran sulfate 3 and 2.6M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 3 was coated at a 2.6M NaCl concentration.

EXAMPLE 1.10 preparation of a coating on PVC pipe Using dextran sulfate 3 and 3.0M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 3 was coated at a concentration of 3.0M NaCl.

EXAMPLE 1.11 preparation of a coating on PVC pipe Using dextran sulfate 4 and 0.05M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 4 was coated at 0.05M NaCl concentration. .

EXAMPLE 1.12 preparation of a coating on PVC pipe Using dextran sulfate 4 and 0.1M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 4 was coated at 0.1M NaCl concentration.

EXAMPLE 1.13 preparation of a coating on PVC pipe Using dextran sulfate 4 and 0.25 MNaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 4 was coated at 0.25M NaCl concentration.

EXAMPLE 1.14 use of dextran sulfate 4 and 1.0M Preparation of coating on PVC pipe by NaCl concentration

EXAMPLE 1.15 preparation of a coating on PVC pipe Using dextran sulfate 4 and 1.7MNaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 4 was coated at a 1.7M NaCl concentration.

EXAMPLE 1.16 use of dextran sulfate 4 and 3.0M Preparation of coating on PVC pipe by NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 4 was coated at a concentration of 3.0M NaCl.

Example 1Preparation of a coating on PVC pipe Using dextran sulfate 5 and MNaCl concentration 0.05

PVC pipes were coated according to the general method described above. Referring to table 1, dextran sulfate 5 was coated at 0.05M NaCl concentration.

EXAMPLE 1.18 use of dextran sulfate 5 and 0.25M Preparation of coating on PVC pipe by NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 5 was coated at 0.25M NaCl concentration.

EXAMPLE 1.19 preparation of a coating on PVC pipe Using dextran sulfate 5 and 0.5M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 5 was coated at 0.5M NaCl concentration.

EXAMPLE 1.20 preparation of a coating on PVC pipe Using dextran sulfate 5 and 0.85MNaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 5 was coated at 0.85M NaCl concentration.

EXAMPLE 1.21 preparation of a coating on PVC pipe Using dextran sulfate 5 and 1.0M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 5 was coated at 0.1M NaCl concentration.

EXAMPLE 1.22 preparation of a coating on PVC pipe Using dextran sulfate 5 and 1.7M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 5 was coated at a 1.7M NaCl concentration.

EXAMPLE 1.23 use of dextran sulfate 5 and 3.0M Preparation of coating on PVC pipe by NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 5 was coated at a concentration of 3.0M NaCl.

EXAMPLE 1.24 dextran sulfate 6 and 0.05MNaCl concentration were usedPreparation of coatings on PVC pipes

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 6 was coated at 0.05M NaCl concentration.

EXAMPLE 1.25 use of dextran sulfate 6 and 0.25M Preparation of coating on PVC pipe by NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 6 was coated at 0.25M NaCl concentration.

EXAMPLE 1.26 preparation of a coating on PVC pipe Using dextran sulfate 6 and 0.5M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 6 was coated at 0.5M NaCl concentration.

EXAMPLE 1.27 use of dextran sulfate 6 and 1.7M Preparation of coating on PVC pipe by NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 6 was coated at a 1.7M NaCl concentration.

EXAMPLE 1.28 preparation of a coating on PVC pipe Using dextran sulfate 6 and 3.0M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 6 was coated at a concentration of 3.0M NaCl.

EXAMPLE 1.29 preparation of a coating on PVC pipe Using dextran sulfate 7 and 0.05MNaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 7 was coated at 0.05M NaCl concentration.

EXAMPLE 1.30 preparation of a coating on PVC pipe Using dextran sulfate 7 and 0.1M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 7 was coated at 0.1M NaCl concentration.

EXAMPLE 1.31 preparation of a coating on PVC pipe Using dextran sulfate 7 and 0.25 MNaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 7 was coated at 0.25M NaCl concentration.

EXAMPLE 1.32 preparation of a coating on PVC pipe Using dextran sulfate 7 and 0.5M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 7 was coated at 0.5M NaCl concentration.

EXAMPLE 1.33 preparation of a coating on PVC pipe Using dextran sulfate 7 and 0.85M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 7 was coated at 0.85M NaCl concentration.

EXAMPLE 1.34 preparation of a coating on PVC pipe Using dextran sulfate 7 and 1.0M NaCl concentration

EXAMPLE 1.35 preparation of a coating on PVC pipe Using dextran sulfate 7 and 1.7M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 7 was coated at a 1.7M NaCl concentration.

EXAMPLE 1.36 preparation of a coating on PVC pipe Using dextran sulfate 7 and 2.6M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 7 was coated at a 2.6M NaCl concentration.

EXAMPLE 1.37 preparation of a coating on PVC pipe Using dextran sulfate 7 and 3.0M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 7 was coated at a concentration of 3.0M NaCl.

EXAMPLE 1.38 preparation of a coating on PVC pipe Using dextran sulfate 7 and 3.4M NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 7 was coated at a concentration of 3.4M NaCl.

₂ ₄ EXAMPLE 1.39 preparation of a coating on PVC pipe using dextran sulfate 5 and NaHPO concentration of 0.05M

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to Table 1, dextran sulfate 5 was treated with 0.05M Na ₂ HPO ₄ Concentration coating.

₂ ₄ EXAMPLE 1.40 preparation of a coating on PVC pipe using dextran sulfate 5 and NaHPO concentration of 0.25M

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to Table 1, dextran sulfate 5 was treated with 0.25M Na ₂ HPO ₄ Concentration coating.

₂ ₄ EXAMPLE 1.41 preparation of a coating on PVC pipe using dextran sulfate 5 and NaHPO concentration of 0.85M

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to Table 1, dextran sulfate 5 was treated with 0.85M Na ₂ HPO ₄ Concentration coating.

₂ ₄ EXAMPLE 1.42 preparation of a coating on PVC pipe using dextran sulfate 5 and NaHPO concentration of 1.7M

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to Table 1, dextran sulfate 5 was treated with 1.7M Na ₂ HPO ₄ Concentration coating

₂ ₄ EXAMPLE 1.43 preparation of a coating on PVC pipe Using dextran sulfate 5 and NaSO concentration of 0.05M

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to Table 1, dextran sulfate 5 was treated with 0.05M Na ₂ SO ₄ Concentration coating.

₂ ₄ EXAMPLE 1.44 preparation of a coating on PVC pipe using dextran sulfate 5 and NaSO concentration of 0.25M

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to Table 1, dextran sulfateSugar 5 at 0.25M Na ₂ SO ₄ Concentration coating.

₂ ₄ EXAMPLE 1.45 preparation of a coating on PVC pipe using dextran sulfate 5 and NaSO concentration of 0.85M

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to Table 1, dextran sulfate 5 was treated with 0.85M Na ₂ SO ₄ Concentration coating.

₂ ₄ EXAMPLE 1.46 preparation of a coating on PVC pipe using dextran sulfate 7 and NaHPO concentration of 0.85M

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to Table 1, dextran sulfate 7 was treated with 0.85M Na ₂ HPO ₄ Concentration coating.

₂ ₄ EXAMPLE 1.47 preparation of a coating on PVC pipe using dextran sulfate 7 and NaSO concentration of 0.85M

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to Table 1, dextran sulfate 7 was treated with 0.85M Na ₂ SO ₄ Concentration coating.

EXAMPLE 1.48 use of dextran sulfate 7 and 0.05M Preparation of coating on PUR tube with NaCl concentration

PUR tubes (i.d. 3 mm) were coated according to the general procedure described above. Referring to table 1, dextran sulfate 7 was coated at 0.05M NaCl concentration.

EXAMPLE 1.49 preparation of a coating on a PUR tube using dextran sulfate 7 and a concentration of 0.25MNaCl

PUR tubes (i.d. 3 mm) were coated according to the general procedure described above. Referring to table 1, dextran sulfate 7 was coated at 0.25M NaCl concentration.

EXAMPLE 1.50 use of dextran sulfate 7 and 1.7M Preparation of coating on PUR tube with NaCl concentration

PUR tubes (i.d. 3 mm) were coated according to the general procedure described above. Referring to table 1, dextran sulfate 7 was coated at a 1.7M NaCl concentration.

EXAMPLE 1.51 use of dextran sulfate 7 and 3.0M NaCl concentrationPreparation of coatings on PUR tubes

PUR tubes (i.d. 3 mm) were coated according to the general procedure described above. Referring to table 1, dextran sulfate 7 was coated at a concentration of 3.0M NaCl.

EXAMPLE 1.52 use of dextran sulfate 7 and 0.05M Coating on steel sampling tube prepared by NaCl concentration

A steel coupon (15.0 mm. Times.3.35 mm) was coated according to the general procedure described above. Referring to table 1, dextran sulfate 7 was coated at 0.05M NaCl concentration.

EXAMPLE 1.53 preparation of coating on Steel coupon Using dextran sulfate 7 and 0.25MNaCl concentration

A steel coupon (15.0 mm. Times.3.35 mm) was coated according to the general procedure described above. Referring to table 1, dextran sulfate 7 was coated at 0.25M NaCl concentration.

EXAMPLE 1.54 preparation of coating on Steel coupon Using dextran sulfate 7 and 1.7MNaCl concentration

A steel coupon (15.0 mm. Times.3.35 mm) was coated according to the general procedure described above. Referring to table 1, dextran sulfate 7 was coated at a 1.7M NaCl concentration.

EXAMPLE 1.55 preparation of coating on Steel coupon Using dextran sulfate 7 and 3.0MNaCl concentration

A steel coupon (15.0 mm. Times.3.35 mm) was coated according to the general procedure described above. Referring to table 1, dextran sulfate 7 was coated at a concentration of 3.0M NaCl.

EXAMPLE 1.56 use of dextran sulfate 2 and 1.7M Preparation of coating on PVC pipe by NaCl concentration

PVC pipes (I.D.3mm) were coated according to the general method described above. Referring to table 1, dextran sulfate 2 was coated at a 1.7M NaCl concentration.

Example 2a normalized heparin Activity of PVC pipes coated with different dextran sulfate at different NaCl concentrations

Heparin activity was determined for PVC tubes coated at different NaCl concentrations according to examples 1.11-1.19,1.21-1.32,1.34-1.38 (corresponding to

dextran sulfate

4, 5, 6 and 7) as described in evaluation method B (heparin activity test).

All coated solid objects tested exhibited at least 1pmol/cm when tested using evaluation method B ² Is a heparin activity of (a). The heparin activity values shown in table 2 below were normalized to the highest heparin activity values observed for PVC tubes coated with dextran sulfate 5 at 1.7M NaCl (example 1.22).

Table 2-normalized heparin activity of PVC pipes coated with different NaCl concentrations using

dextran sulfate

4, 5, 6 and 7 (％)

FIG. 2 shows normalized heparin activity values of PVC tubing coated with

dextran sulfate

4, 5, 6 and 7 at 0.25M and 1.7M NaCl. As can be seen from fig. 2, the use of a higher salt concentration (1.7M) in the step of adding the dextran sulfate layer resulted in higher heparin activity than the use of a lower salt concentration (0.25M), although both salt concentrations resulted in a coating with acceptable antithrombotic properties. As can be seen from Table 2, the use of salt concentrations less than 0.25M reduced heparin activity. The highest heparin activity was obtained with dextran sulfate (

dextran sulfate

4, 5, 7) having a charge density higher than 6. Mu. Eq/g.

EXAMPLE 2b normalized heparin Activity of coated PVC tubing with dextran sulfate 5 with different salts at different concentrations

As described in evaluation method B (heparin Activity test), the assay uses different concentrations of NaCl, na ₂ HPO ₄ Or Na (or) ₂ SO ₄ Heparin activity of PVC pipes coated according to examples 1.17, 1.18, 1.20, 1.22 and 1.39-1.45 (corresponding to dextran sulfate 5).

All the coated solid objects tested exhibited at least 1pmol/cm ² Is a heparin activity of (a). The heparin activity values shown in table 3 below were normalized to the highest heparin activity observed for PVC tubes coated with dextran sulfate 5 at 1.70M NaCl (example 1.22).

TABLE 3 standardized heparin Activity of PVC tubes (dextran sulfate 5) coated with different salts at different concentrations Sex (%)

* Insoluble in water at 1.7M

Normalized heparin activity values from table 3 are shown in figure 3. As can be seen from fig. 3, the beneficial effect of using higher salt concentrations on heparin activity in the step of adding the dextran sulfate layer is shown by a series of salts. The highest heparin activity value was obtained using sodium chloride.

EXAMPLE 2c normalized heparin Activity of coated PVC tubing with

dextran sulfate

5 and 7 with different salts at 0.85M concentration

Determination according to evaluation method B (heparin Activity test) according to examples 1.20, 1.33, 1.41 and 1.45-1.47 (corresponding to dextran sulfate 5 and 7), 0.85M NaCl, na were used ₂ HPO ₄ Or Na (or) ₂ SO ₄ Heparin activity of coated PVC tubing.

All the coated solid objects tested exhibited at least 1pmol/cm ² Is a heparin activity of (a). The heparin activity values shown in table 4 below were normalized according to the highest heparin activity value observed in example 1.22.

Table 4-normalized heparin activity of PVC tubes (dextran sulfate 5 and 7) coated with different salts at 0.85M concentration (％)

It can be seen that different salts such as NaCl, na are used ₂ HPO ₄ And Na (Na) ₂ SO ₄ The heparin activity value is not significantly affected. Whichever salt is used, the salt concentration will affect heparin activity.

EXAMPLE 2d normalized heparin Activity of solid bodies coated with dextran sulfate 7 with different concentrations of NaCl

The heparin activity of the various coated solid bodies according to examples 1.29, 1.31, 1.35, 1.37 and 1.48-1.55 (corresponding to dextran sulfate 7) using different concentrations of NaCl was determined as described in evaluation method B (heparin activity test).

All the coated solid objects tested exhibited at least 1pmol/cm ² Is a heparin activity of (a). The heparin activity values shown in table 5 below were normalized according to the highest heparin activity value observed in example 1.22.

Table 5- (dextran sulfate 7) normalized heparin activity with different coated solid bodies of different concentrations of NaCl (％)

Examples numbering	Dextran sulfate numbering	Salt concentration [ M ]]	PVC	PUR	Steel product
						1.29/1.48/1.52	7	0.05	46	40	65
1.31/1.49/1.53	7	0.25	60	62	81
						1.35/1.50/1.54	7	1.70	98	110	135
1.37/1.51/1.55	7	3.00	64	72	88

It is evident from table 5 that the salt concentration influences the activity of heparin, irrespective of the material of the coated solid object. Dextran sulfate 7 was coated at different salt concentrations for Polyurethane (PUR) made tubes and steel sampling tubes, and the standardized heparin activity values obtained indicated the presence of a significant salt dependence.

Example 2e normalized heparin Activity of PVC tubes coated with dextran sulfate 5 and heparin fragment (octasaccharide) at 1.7M NaCl concentration

Heparin fragments (octasaccharides) were coated with dextran sulfate 5 at 1.7M NaCl concentration on PVC tubing (I.D.3 mm) according to the general procedure described above, see Table 1.

Heparin fragment fractions prepared by heparin depolymerization and then fractionation

Oligosaccharides of predominantly eight sugar unit (octasaccharide) size can be prepared by partial nitrous acid cleavage of natural heparin followed by fractionation by gel chromatography. The octasaccharide produced by nitrous acid cleavage is the shortest fragment capable of containing a functionally active sequence (Thunberg L. Et al, FEBS Letters 117 (1980), 203-206).

Depolymerization of heparin 10g of heparin sodium were dissolved in 36ml of water by stirring overnight. 0.30g NaNO ₂ Added to heparin solution and allowed to dissolve. The solution was acidified to pH 2.5 by addition of 4M HCl. After a total reaction time of 2h at room temperature, the solution was neutralized by adding 4M NaOH.

The degradation mixture was separated on the basis of molecular size by Gel Permeation Chromatography (GPC), wherein 3ml fractions were loaded onto a column (HiLoad 26/600Superdex 30pg, mobile phase 0.15M NaCl) at a flow rate of 2.5 ml/min. The fractions (3 ml) collected were analyzed for aldehydes by MBTH reaction essentially as described in Smith r.l. and Gilkerson E (1979), anal Biochem 98, 478-480. A broad peak centered at the octasaccharide elution position was collected. Pooled oligosaccharide elution fractions from several preparative runs were concentrated by evaporation to a volume of 18ml and subjected to further chromatographic separation on the same column. For all the re-chromatographic runs, three fractions representing the decasaccharide, octasaccharide and hexasaccharide fragments were collected and pooled.

The collected components were analyzed by evaluation method P. The "hexa" component consists of a major peak representing hexasaccharide and a shoulder representing octasaccharide. The "eight" component consists of a major peak representing octasaccharide and a minor shoulder representing hexasaccharide and a minor shoulder representing decasaccharide. The "ten" component consisted of a major peak representing ten sugars and a shoulder representing eight sugars with a small shoulder representing twelve sugars.

The concentration of the heparin fragment component was determined by evaluation method Q (see table below).

Fixation of octasaccharide

The PVC tube was coated with 16ml of "eight" component diluted with 84ml of 0.05M NaCl, and then the eight component was immobilized on the outermost layer of the polyamine by reductive amination, essentially as described in Larm et al, EP 008686A 1 and EP0495820B1 (both incorporated herein by reference in their entirety).

Evaluation of toluidine staining of PVC tubing coated with heparin fragments

The oligosaccharide coated surfaces were subjected to the toluidine blue staining test as described in evaluation method C. Intense blue/purple color was observed on the luminal surface of the tube, indicating extensive covalent binding of heparin fragments. The uniform staining obtained for the test tube indicated that a uniform coating was formed.

Heparin Density evaluation Using heparin fragment coated PVC tubes

The surface heparin density was determined by evaluation method a and the results are shown in the following table.

Evaluation of heparin Activity of PVC tubes coated with heparin fragments

The heparin activity of the octasaccharide coated surface (example 2 e) was determined by evaluation method B. The heparin activity values shown in the table below were normalized to the highest heparin activity observed for the coated PVC tube (example 1.22) with dextran sulfate 5 at 1.70M NaCl.

Examples numbering	Heparin Density (μg/cm) ² )	Heparin Activity (%)
			2e	5.6	5
1.22	6.5	100

Although the heparin density values of the octasaccharide coating (example 2 e) and the heparin coating (example 1.22) were similar, the AT binding capacity (heparin activity; 'HA') of the octasaccharide coating was lower than that of the heparin coating. However, this is expected given the relatively low anti-FXa activity exhibited by the octasaccharide component in solution (data not shown). Thus, the octasaccharide fragment appears to substantially retain its AT binding capacity after immobilization.

Example 3a heparin concentration of PVC tubes coated with different dextran sulfate at different NaCl concentrations

Heparin concentrations were determined for solid objects (PVC pipes) coated with different NaCl concentrations according to examples 1.4-1.19,1.21-1.32 and 1.34-1.38 (corresponding to

dextran sulfate

3, 4, 5, 6 and 7) as described in evaluation method a.

The coated solid objects all tested exhibited at least 1 μg/cm ² Heparin concentration of (c). Heparin concentration values are shown in table 6 below.

TABLE 6 heparin concentration (μg +. ² cm)

FIG. 4 shows heparin concentration of PVC tubing coated with

dextran sulfate

3, 4, 5, 6 and 7 at 1.7M NaCl. As can be seen from fig. 4, under these conditions, there is a trend towards higher heparin concentrations using higher molecular weight dextran sulfate in the step of adding the dextran sulfate layer. In this example, dextran sulfate 3 is the reference dextran sulfate.

Figure 5 shows heparin concentrations of PVC pipes coated with

dextran sulfate

3, 4, 5, 6 and 7 at different NaCl concentrations. As can be seen from fig. 5, during the step of adding the dextran sulfate layer,

dextran sulfate

4, 5, 6 and 7 showed a tendency of increasing heparin concentration with increasing salt concentration (at least up to 1.7M). It can be seen that the use of salt concentrations less than 0.25M generally results in lower heparin activity. Dextran sulfate 3 does not follow this trend and when it is used, the heparin concentration decreases with increasing salt concentration in this step. In this example, dextran sulfate 3 is a reference dextran sulfate. Without being limited by theory, the inventors attribute this trend difference to the fact that: dextran sulfate 3 has a much lower charge density than

dextran sulfate

4, 5, 6 and 7.

Example 3b heparin concentration of PVC pipe coated with dextran sulfate 5 with different salts at different concentrations

As described in evaluation method A, the assays according to examples 1.17, 1.18, 1.20, 1.22 and 1.39-1.45 (corresponding to dextran sulfate 5) were carried out using NaCl, na ₂ HPO ₄ Or Na (or) ₂ SO ₄ Heparin concentration of PVC pipes coated at different concentrations.

The coated solid objects all tested exhibited at least 1 μg/cm ² Heparin concentration of (c). Heparin concentration values are shown in table 7 below.

² TABLE 7 heparin concentration (. Mu.g/cm) of PVC tubes coated with different salts at different concentrations (dextran sulfate 5)

/>

*Na ₂ SO ₄ Insoluble in water at 1.7M

Heparin concentration values in table 7 are shown in fig. 6. As can be seen from fig. 6, dextran sulfate 5 shows a tendency to increase heparin concentration with increasing salt concentration during the step of adding the dextran sulfate layer for a range of different salts.

Example 3c heparin concentration of PVC pipe coated with

dextran sulfate

5 and 7 with different salts at 0.85M concentration

According to the evaluation method A, the determination was carried out according to examples 1.20, 1.33, 1.41, 1.45, 1.46 and 1.47 (corresponding to dextran sulfate 5 and 7), using NaCl, na ₂ HPO ₄ Or Na (or) ₂ SO ₄ Heparin concentration of 0.85M coated PVC tubing.

The coated solid objects all tested exhibited at least 1 μg/cm ² Heparin concentration of (c). Heparin concentration values are shown in table 8 below.

TABLE 8 heparin concentration (μg +. ² cm)

Examples numbering	Dextran sulfate numbering	Salt concentration [ M ]]	Na ₂ HPO ₄	Na ₂ SO ₄	NaCl
						1.41/1.45/1.20	5	0.85	5.0	4.2	3.5
1.46/1.47/1.33	7	0.85	3.3	3.6	3.7

It can be seen that the different salts such as NaCl, na ₂ HPO ₄ And Na (Na) ₂ SO ₄ The application of (3) does not significantly affect the heparin concentration value.

Example 3d heparin concentration of different coated solid objects with dextran sulfate 7 with different concentrations of NaCl

Heparin concentrations were determined for various solid objects according to examples 1.29, 1.31, 1.35, 1.37 and 1.48-1.55 (corresponding to dextran sulfate 7 (12)) coated with different concentrations of NaCl as described in evaluation method a.

All coated solid objects tested showed at least 1. Mu.g/cm ² Heparin concentration of (c). Heparin concentration values are shown in table 9 below.

TABLE 9 heparin concentration (. Mu.g +.A) of different coated solid objects (dextran sulfate 7) using different concentrations of NaCl ² cm)

Examples numbering	Dextran sulfate numbering	Salt concentration [ M ]]	PVC	PUR	Steel product
						1.29/1.48/1.52	7	0.05	4.0	2.9	7.2
1.31/1.49/1.53	7	0.25	5.7	2.5	7.7
						1.35/1.50/1.54	7	1.70	6.8	3.5	10.4
1.37/1.51/1.55	7	3.00	7.3	4.1	8.9

As is evident from table 9, the salt concentration affects the heparin concentration independently of the material of the coated solid object. Both the Polyurethane (PUR) tube and the steel sampling tube were coated with dextran sulfate 7 at different salt concentrations, and the resulting heparin concentration values indicated the presence of a significant salt dependence.

Example 4a zeta potential measurement of PVC pipes coated with different dextran sulphate at 1.7M and 0.25M NaCl concentrations

The surface charges of PVC pipes coated with different NaCl concentrations according to examples 1.1, 1.2, 1.3, 1.6, 1.8, 1.13, 1.15, 1.18, 1.22, 1.25, 1.27, 1.31, 1.35 and 1.56 (corresponding to

dextran sulfate

1, 2, 3, 4, 5, 6 and 7) were determined as described in evaluation method D.

Zeta potential values of PVC tubes coated with dextran sulfate 1-7 at 1.7M NaCl are shown in Table 10.

TABLE 10 zeta potential of PVC pipes coated with dextran sulfate 1-7 with 1.7M NaCl

Dextran sulfate 1-7 all have IEP below pH 3. However, the lower molecular weight dextran sulfate (reference examples dextran sulfate 1-3) did not achieve all of the preferred features (i.e., potential fingerprints of the solid objects coated according to the method of the present invention described above).

Dextran sulfate

1 and 2 have overall minima (overall minima) that occur at pH above 5, and dextran sulfate 3 has a delta value below 20 mV. The solid objects of the invention coated with dextran sulfate 4-7 meet these criteria.

Zeta potentials of PVC pipes coated with

dextran sulfate

3, 4 and 5 at 1.7M NaCl (corresponding to examples 1.8, 1.15 and 1.22) are shown in fig. 7.

Zeta potentials of PVC pipes coated with

dextran sulfate

3, 6 and 7 at 1.7M NaCl (corresponding to examples 1.8, 1.27 and 1.35) are shown in fig. 8.

Zeta potential values of PVC tubes coated with dextran sulfate 1-7 at 0.25M NaCl are shown in Table 11.

Table 11-dextran sulfate 1-7 was used at 0.Zeta potential of PVC pipe coated with 25M NaCl

Dextran sulfate 1-7 all have IEP below pH 3. However, the lower molecular weight dextran sulfate (reference examples dextran sulfate 1-3) did not meet all of the preferred characteristics (i.e., potential fingerprints of the solid objects coated according to the method of the present invention described above). Dextran sulfate 2 has an overall minimum that occurs at a pH above 5, while

dextran sulfate

1 and 3 have a delta value below 20 mV. The solid objects of the invention coated with dextran sulfate 4 to 7 meet these criteria.

Zeta potentials of PVC pipes (corresponding to examples 1.6, 1.13 and 1.18) coated with

dextran sulfate

3, 4 and 5 at 0.25M NaCl are shown in fig. 9.

Zeta potentials of PVC pipes (corresponding to examples 1.6, 1.25 and 1.31) coated with

dextran sulfate

3, 6 and 7 at 0.25M NaCl are shown in fig. 10.

Example 4b measurement of zeta potential of PVC pipes coated with dextran sulfate 5 at different concentrations of different salts

As described in evaluation method D, the determination was performed using different concentrations of NaCl, na ₂ HPO ₄ Or Na (or) ₂ SO ₄ According to examples 1.18, 1.22, 1.39 to 1.42 and 1.44 to 1.45 (all dextran sulfate 5).

The zeta potential of PVC pipes coated with dextran sulfate 5 at different NaCl concentrations is shown in table 12.

TABLE 12 zeta potential of PVC pipes coated with dextran sulfate 5 at different NaCl concentrations

Examples numbering	Salt concentration [ M ]]	Delta value [ mV]	PH (overall minimum)	IEP
					1.18	0.25	38	4.5	2.4
1.22	1.7	28	4.5	2.5

Figure 11 shows the zeta potential spectra of PVC pipes coated with dextran sulfate 5 at 0.25M and 1.7M NaCl concentrations (corresponding to examples 1.18 and 1.22), where the effect of the salt on the zeta potential is evident. Preferred characteristics (i.e. potential fingerprints of the solid objects coated according to the method of the invention described above) are met at 0.25 and 1.7M NaCl concentrations.

Dextran sulfate 5 in different Na ₂ HPO ₄ Zeta potential values of coated PVC pipes at concentrations are shown in table 13.

₂ ₄ TABLE 13 zeta potential of PVC pipes coated with dextran sulfate 5 at different NaHPO concentrations

FIG. 12 showsDextran sulfate 5 at 0.25M, 0.85M and 1.7M Na ₂ HPO ₄ Zeta potential spectra of concentration-coated PVC pipes (corresponding to examples 1.40, 1.41 and 1.42), in which it can be observed that different concentrations of Na are used ₂ HPO ₄ All the preferred features (i.e. the potential fingerprint of the solid object coated according to the method of the invention described above) are met.

Dextran sulfate 5 was used with different Na ₂ SO ₄ Zeta potential values of the concentration-coated PVC pipes are shown in table 14.

₂ ₄ TABLE 14 zeta potential of PVC pipes coated with dextran sulfate 5 at different NaSO concentrations

Examples numbering	Salt concentration [ M ]]	Delta value [ mV]	PH (overall minimum)	IEP
					1.44	0.25	40	4.5	2.6
1.45	0.85	39	4.3	2.6
					N/A**	1.7	*	*	*

* Insoluble in water at 1.7M

* N/a = inapplicable

FIG. 13 shows the use of dextran sulfate 5 with Na ₂ SO ₄ Zeta potential spectra of 0.25M and 0.85M coated PVC pipes (corresponding to examples 1.44 and 1.45), where it can be seen that different concentrations of Na are used ₂ SO ₄ All the preferred features (i.e. the potential fingerprint of a solid object coated according to the method of the invention described above) are met. It can be seen from tables 12, 13 and 14 that the zeta potential spectra are not significantly affected by the use of different types of salts at different concentrations. It is also clear that there is also salt dependence for different salt types.

Example 5 blood contact activation (platelet loss and F1+2) of PVC tubes coated with different dextran sulfate at different NaCl concentrations

The percent of platelets and f1+2 (prothrombin fragments) saved after exposure of PVC tubes coated with different NaCl concentrations according to examples 1.1, 1.3, 1.13, 1.18, 1.25 and 1.31 (corresponding to

dextran sulfate

1, 2, 4, 5, 6 and 7) to blood was determined as described in evaluation methods E and F, respectively.

The results are shown in Table 15 and FIGS. 14 and 15 (0.25M NaCl concentration) and Table 16 and FIGS. 16 and 17 (1.7M NaCl concentration).

As can be seen from these tables and figures, no significant platelet loss (platelet loss indicating thrombosis) was observed for solid objects coated with

dextran sulfate

4, 5, 6 and 7 according to the method of the present invention at 0.25M and 1.7M NaCl concentrations. The antithrombotic properties of the coating were further confirmed by the low f1+2 values (prothrombin fragments) observed for the same dextran sulfate. The tubes coated with comparative dextran sulfate 1 having a molecular weight of 50kDa and comparative dextran sulfate 2 having a molecular weight of 100kDa also showed significant thrombosis and high formation of prothrombin fragments compared to the solid bodies coated with dextran sulfate 4-7 of the present invention.

Uncoated PVC tubing and coagulation examples showed significant thrombosis in this experiment.

TABLE 15 preserved blood platelets from PVC tubes coated with

dextran sulfate

1, 2, 4, 5, 6 and 7 at 0.25M NaCl concentration Plates (%) and F1+2 (pmol/L)

TABLE 16 preservation of platelets using

dextran sulfate

1, 4, 5, 6 and 7 PVC tubes coated at 1.7M NaCl concentration (%) and F1+2 (pmol/L)

Example 6: toluidine staining of PVC and PUR tubes and steel sampling tubes coated with different dextran sulfate at different salt concentrations

PVC and PUR tubes and steel sampling tubes coated according to examples 1.1-1.55 were subjected to the toluidine blue staining test as described in evaluation method C.

Blue/purple color was observed on the luminal surface of the tube and steel sampling tube, indicating covalent attachment of end-functionalized heparin. For the uniform staining obtained for the solid objects coated according to the method of the invention tested, it was shown that a uniform coating (in particular a uniform heparin distribution) was formed, which can be obtained on different solid objects with different concentrations of salts using different dextran sulfate.

Example 7 blood contact activation (platelet loss and F1+2) of coated PVC tubes after temperature and humidity testing

PVC tubes coated with different NaCl concentrations according to examples 1.13, 1.15, 1.22 and 1.35 (corresponding to

dextran sulfate

4, 5 and 7) were exposed to elevated temperature and relative humidity (40 ℃,50% rh for 1 week, according to evaluation method K) and then evaluated according to evaluation methods E (preserved platelets) and F (f1+2). The results are shown in table 17 and fig. 18 and 19, and table 18 and fig. 20 and 21.

As shown in these tables and figures, there was no significant change in the platelets and f1+2 values stored after exposure to elevated temperature and humidity for the solid objects coated with

dextran sulfate

4, 5 and 7 according to the method of the present invention. Solid objects coated with

dextran sulfate

4, 5 and 7 according to the method of the present invention prepared at 0.25M and 1.7M NaCl concentrations gave similar results.

These results show that the antithrombotic properties of the coated solid objects prepared according to the method of the invention are retained despite exposure to severe conditions, such as elevated temperature and humidity.

TABLE 17 preserved platelets (%) and F1 of PVC tubes coated with dextran sulfate 4 at 0.25M NaCl concentration +2 (pmol/L) -before and after exposure to elevated temperature and humidity

TABLE 18 use of

dextran sulfate

4, 5 and 7 at 1.7M Preservation of NaCl-coated PVC pipePlatelets (%) and f1+2 (pmol/L) -before and after exposure to elevated temperature and humidity

All patents and patent applications cited herein are incorporated by reference in their entirety.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer, step, group of integers or steps but not the exclusion of any other integer, step, group of integers or group of steps.

Embodiments are described below:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

2. The method for producing a solid object according to embodiment 1, wherein the anionic polymer is dextran sulfate.

3. The method for producing a solid object according to embodiment 1 or embodiment 2, wherein the anionic polymer is characterized by having a total molecular weight of 750kDa to 10,000kDa, e.g. 1,000kDa to 10,000kDa.

4. The method for producing a solid object according to any one of embodiments 1 to 3, wherein the total molecular weight of the anionic polymer is determined according to evaluation method G.

5. The method for producing a solid object according to any one of embodiments 1 to 4, wherein the anionic polymer is characterized by having a solution charge density of from >4 to 7 μeq/g, for example 5 to 7 μeq/g.

6. The process for preparing a solid object according to any of embodiments 1-5, wherein step ii) is performed at a salt concentration of 0.25M-4.0M, e.g. 0.25M-3.0M.

7. The method for producing a solid object according to any one of embodiments 1 to 6, wherein the salt is an inorganic salt.

8. The method for producing a solid object according to embodiment 7, wherein the salt is an inorganic sodium salt.

9. The method for producing a solid object according to embodiment 8, wherein the salt is selected from the group consisting of sodium chloride, sodium sulfate, sodium hydrogen phosphate, and sodium phosphate.

10. The method for producing a solid object of embodiment 9, wherein the salt is sodium chloride.

11. The process for preparing a solid object according to any of embodiments 1-10, wherein step iii) is not optional.

12. The method for producing a solid object according to embodiment 11, wherein in step iii), steps i) and ii) are repeated 1 to 10 times, for example 1, 2, 3, 4, 5 or 6 times.

13. The process for preparing a solid object according to any of embodiments 1 to 12, wherein the cationic polymer of step i) is the same as the cationic polymer of step iv).

14. The process for preparing a solid object according to any of embodiments 1-13, wherein the cationic polymer of step i) is a polyamine, which is optionally crosslinked.

15. The process for preparing a solid object according to any of embodiments 1-14, wherein the cationic polymer of step iv) is a polyamine, which is optionally crosslinked.

16. The method for producing a solid object according to any one of embodiments 1 to 15, further comprising a pretreatment step before step i).

17. The method for producing a solid object according to any one of embodiments 1 to 16, further comprising a step between step i) and step ii), step ii) and step iii), step iii) and step iv), or step iv) and step v).

18. The method for producing a solid object according to any one of embodiments 1 to 17, wherein the anticoagulant substance is a heparin moiety.

19. The method for preparing a solid object of embodiment 18, wherein the heparin moiety is a terminally linked heparin moiety.

20. The method for producing a solid object according to embodiment 19, wherein the terminally linked heparin moiety is linked by its reducing end.

21. The method for preparing a solid object of embodiment 18, wherein the anticoagulant substance is full length heparin.

22. The method for preparing a solid object according to any of embodiments 1-21, wherein the anticoagulant substance is covalently linked through a linking group.

23. The method for preparing a solid object of embodiment 22, wherein the linking group comprises a secondary amine.

24. The method for preparing a solid object of embodiment 22, wherein the linking group comprises a secondary amide.

25. The method for preparing a solid object of embodiment 22, wherein the linking group comprises 1,2, 3-triazole.

26. The method for preparing a solid object of embodiment 22, wherein the linking group comprises a thioether.

27. The method for producing a solid object according to any one of embodiments 1 to 26, wherein the solid object is a medical device, an analysis device, a separation device, or a membrane.

28. The method for preparing a solid object of embodiment 27, wherein the solid object is an antithrombotic medical device.

29. The method for preparing a solid object of embodiment 27, wherein the solid object is an extracorporeal medical apparatus.

30. The method for preparing a solid object of embodiment 28, wherein the solid object is an in vivo medical device.

31. The method for preparing a solid object of embodiment 30, wherein the in vivo medical device is a stent or stent-graft.

32. The method for producing a solid object according to any one of embodiments 1 to 31, wherein the solid object has at least 1pmol/cm ² Anticoagulant activity for binding to the surface of ATIII, e.g. at least 2pmol/cm ² Surface for binding ATIII, at least 3pmol/cm ² Surface for binding ATIII, at least 4pmol/cm ² For binding to surfaces of ATIII or at least 5pmol/cm ² For binding to the surface of ATIII, suitably it is determined according to evaluation method B.

33. The method for preparing a solid object according to any of embodiments 1-32, wherein the solid object has a blood contacting property of at least 80% preserved platelets, e.g. at least 85% preserved platelets, e.g. at least 90% preserved platelets, suitably determined according to evaluation method E.

34. The method for producing a solid object according to any one of embodiments 1 to 33, wherein the solid object has an f1+2 value of <10,000pmol/L, less than 7,500pmol/L, less than 5,000pmol/L or less than 4,000pmol/L, suitably it is determined according to evaluation method F.

35. The method for producing a solid object according to any one of embodiments 1 to 34, wherein the anticoagulant substance is a heparin fraction, and wherein the solid object has at least 1 μg/cm ² For example at least 2. Mu.g/cm ² At least 4. Mu.g/cm ² At least 5 μg/cm ² Or at least 6. Mu.g/cm ² Suitably, it is determined according to evaluation method a.

36. The method for producing a solid object according to any one of embodiments 1 to 35, wherein the solid object has a zeta potential profile, suitably determined according to evaluation method D, characterized by an isoelectric point (IEP) below pH 3, an overall minimum of the curve below pH 5, and a delta value, i.e. the difference between the zeta potential at the overall minimum and the zeta potential at pH9, of at least 20mV.

37. Any one of embodiments 1 through 36The process for producing a solid object according to, wherein the anionic polymer is a polymer comprising a polymer selected from the group consisting of-CO ₂ ^- 、-SO ₃ ^- 、-PO ₃ H ^- and-PO ₃ ^2- Is a polymer of groups of (a).

38. The method for producing a solid object of embodiment 37, wherein the anionic polymer is a polymer comprising-SO ₃ ^- Polymers of groups.

39. The method for preparing a solid object of embodiment 38, wherein the sulfur content of the anionic polymer is from 10% to 25% by weight of the anionic polymer.

40. A method for preparing a solid object having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating comprises an anticoagulant substance, the method comprising the steps of:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

And wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

41. A method for preparing a solid object having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating comprises an anticoagulant substance, the method comprising the steps of:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

the anionic polymer is dextran sulfate;

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

42. The method for producing a solid object according to embodiment 40 or 41, wherein the anticoagulant substance is a heparin moiety.

43. A solid object having a surface comprising a layered coating of cationic and anionic polymers, wherein the outer coating is a layer comprising a cationic polymer covalently linked to an anticoagulant substance;

44. The solid object of embodiment 43, wherein the anionic polymer is characterized by having a total molecular weight of 650kDa to 1,000 kDa.

45. The solid object of embodiment 43, wherein the anionic polymer is characterized by having a total molecular weight of 1,000kDa to 4,500 kDa.

46. The solid object of embodiment 43, wherein the anionic polymer is characterized by having a total molecular weight of 4,500kDa to 7,000 kDa.

47. The solid object of embodiment 43, wherein the anionic polymer is characterized by having a total molecular weight of 7,000kda to 10,000 kda.

48. The solid object of any of embodiments 43-47, wherein the anionic polymer is applied to the surface at a salt concentration of 0.25M-5.0M, e.g., 0.25M-4.0M or 0.25M-3.0M.

49. The solid object of embodiment 48, wherein the anionic polymer is selected from the group consisting of-CO ₂ ^- 、-SO ₃ ^- 、-PO ₃ H ^- and-PO ₃ ^2- Is a polymer of groups of (a).

50. The solid object of embodiment 49, wherein the anionic polymer is a polymer comprising-SO ₃ ^- Polymers of groups.

51. The solid object of embodiment 50, wherein the sulfur content of the anionic polymer is from 10% to 25% by weight of the anionic polymer.

52. A solid object having a surface comprising a layered coating of cationic and anionic polymers, wherein the outer coating is a layer comprising a cationic polymer covalently linked to an anticoagulant substance; the anionic polymer is a polymer comprising-SO ₃ ^- A polymer of groups, and wherein the anionic polymer is characterized as having: (a) a total molecular weight of 650kDa to 10,000 kDa; and (b) a sulfur content of 10% to 25% by weight of the anionic polymer.

53. A method for preparing a solid object having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating is a layer comprising a cationic polymer, the method comprising the steps of:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times; and

iv) treating the surface with a cationic polymer;

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

54. A method for preparing a solid object having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating is a layer comprising a cationic polymer, the method comprising the steps of:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times; and

iv) treating the surface with a cationic polymer;

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

55. A method for preparing a solid object having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating is a layer comprising a cationic polymer, the method comprising the steps of:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times; and

iv) treating the surface with a cationic polymer;

the anionic polymer is dextran sulfate;

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

56. A solid object having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating is a layer comprising a cationic polymer;

57. A solid object having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating is a layer comprising a cationic polymer; the anionic polymer is a polymer comprising-SO ₃ ^- A polymer of groups, and wherein the anionic polymer is characterized as having: (a) a total molecular weight of 650kDa to 10,000 kDa; and (b) a sulfur content of 10% to 25% by weight of the anionic polymer.

58. A method for preparing a solid object having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating is a layer comprising an anionic polymer, the method comprising the steps of:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

And wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

59. A method for preparing a solid object having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating is a layer comprising an anionic polymer, the method comprising the steps of:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

60. A method for preparing a solid object having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating is a layer comprising an anionic polymer, the method comprising the steps of:

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

the anionic polymer is dextran sulfate;

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

61. A solid object having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating is a layer comprising an anionic polymer; and wherein the anionic polymer is characterized by having: (a) a total molecular weight of 650kDa to 10,000 kDa; and (b) a solution charge density of > 4. Mu. Eq/g.

62. A solid object having a surface comprising a layered coating of a cationic and an anionic polymer, wherein the outer coating is a layer comprising an anionic polymer; the anionic polymer is a polymer comprising-SO ₃ ^- A polymer of groups, and wherein the anionic polymer is characterized as having: (a) a total molecular weight of 650kDa to 10,000 kDa; and (b) a sulfur content of 10% to 25% by weight of the anionic polymer.

Claims

i) Treating the surface of the solid object with a cationic polymer;

ii) treating the surface with an anionic polymer;

iii) Optionally repeating steps i) and ii) one or more times;

iv) treating the surface with a cationic polymer; and

and wherein the first and second heat exchangers are configured to,

step ii) is carried out at a salt concentration of 0.25M to 5.0M.

2. The method for producing a solid object according to claim 1, wherein the anionic polymer is dextran sulfate.

3. The method for preparing a solid object according to claim 1 or claim 2, wherein the anionic polymer is characterized by having a total molecular weight of 750kDa to 10,000kDa, such as 1,000kDa to 10,000kDa.

4. A method for producing a solid object according to any one of claims 1 to 3, wherein the total molecular weight of the anionic polymer is determined according to evaluation method G.

5. The method for producing a solid object according to any one of claims 1-4, wherein the anionic polymer is characterized by having a solution charge density of from >4 to 7 μeq/g, such as 5 to 7 μeq/g.

6. The process for preparing a solid object according to any one of claims 1 to 5, wherein step ii) is performed at a salt concentration of 0.25M to 4.0M, such as 0.25M to 3.0M.

7. The method for producing a solid object according to any one of claims 1 to 6, wherein the salt is an inorganic salt.

8. The process for preparing a solid object according to claim 7, wherein the salt is an inorganic sodium salt.

9. The method for producing a solid object according to claim 8, wherein the salt is selected from the group consisting of sodium chloride, sodium sulfate, sodium hydrogen phosphate and sodium phosphate.

10. The method for producing a solid object according to claim 9, wherein the salt is sodium chloride.